Compounds and methods of use to treat infectious diseases

ABSTRACT

The present invention concerns alkyl aryl carbonyl compounds that possess anti-infective activity. The compounds of the invention can be used to target specific nuclear localization signal, thereby blocking importation of specific proteins or molecular complex into the nucleus of a cell. The invention encompasses methods of use of such compounds for treatment or prevention of infectious diseases, such as parasitic and viral diseases, including, for example, malaria and acquired immunodeficiency syndrome. The use of the compounds to detect certain specific protein structures which are present in nuclear localization sequences is also taught.

1 FIELD OF THE INVENTION

The field of the present invention concerns compounds that react withspecific sequences in proteins. The present invention more particularlyconcerns a class of compounds that react, under physiologic conditions,with proteins having adjacent or neighboring basic amino acid sequences.The compounds of the invention can be used to label specifically suchproteins for research purposes and to disrupt their function forpharmacologic purposes. The compounds of the invention can be used fortargeted inactivation of nuclear localization signal in specificproteins or molecular complexes. The compounds of the invention can alsobe used to treat infectious diseases such as HIV infection and malaria.

2 BACKGROUND TO THE INVENTION 2.1 The Derivatization of Proteins

Those skilled in the art will appreciate that there are many compoundsthat can react with specific amino acid residues in proteins, e.g., withsulfhydryl, amino, carboxyl moieties. These reagents are substratespecific, in the sense that each reacts only with one or a few specificamino acids wherever they occur within a protein's sequence. However,the reactivity of such reagents is not affected by the adjacent orneighboring amino acids that form the environment of the reactivemoiety. Thus, the reactivity of such compounds is not context orneighborhood specific.

2.2 Nuclear Importation

The function of an intracellular protein is usually the result of theoverall three dimensional (tertiary) structure of the protein. However,nuclear importation is determined by the simple presence of a shortsequence, called a nuclear localization signal (NLS), which functionsrelatively independently of its position relative to the remainder ofthe structure of object that is imported. In eukaryotic cells allproteins are made in the cytoplasm, which is outside of the nucleus. Ingeneral, those proteins larger than 40 kD that are specificallylocalized in the nucleus of the cell must be actively imported into thenucleus through the nuclear membrane from the cytoplasm via anATP-dependent mechanism that is independent of cell division. Theproteins, and other objects, that are imported have a nuclearlocalization signal (NLS), usually located within the NH₂ terminalsegment of the protein. Several such sequences are known:

a. PKKKRKV from large T antigen of SV40 and other papillomaviruses suchas JC, see Kalderon, D., et al., 1984, Cell 39:499-509;

b. [AV]KRPAATKKAGQAKKKK[LD] from nucleoplasmin, in which only one of thetwo bracketed sequences is required, Dingwall, C., et al., 1988, J. CellBiol. 107:841-49;

c. PRRRRSQS from hepatitis B HbcAg- Yeh, C. T., 1990, J. Virol.

d. KRSAEGGNPPKPLKKLR from the retinoblastoma gene productp110^(rb1)—Zacksenhaus E. et al., 1993, Mol. Cell. Biol. 13:4588

e. KIRLPRGGKKKYKLK from the matrix protein of HIV-1, Bukrinsky, M. I.,et al., 1993, Nature 365:666.

Other viruses that contain NLS sequences include influenza virus (NP,PA, PB1, PB2 proteins which have lysine-rich NLS similar to SV40),hepatitis delta virus (HDAg, which has the sequence PKKKXKK),parvoviruses such as RA1 (NS, VP proteins which have lysine-rich NLSsimilar to SV40), Herpes simplex and measles virus. The recognition ofan NLS sequence is largely independent of the detailed structure of theobject which includes it and of its site of attachment. Goldfarb, D. S.et al., 1986, Nature 332:641-44; Lanford, R. E., 1986, Cell 46:575. Merejuxtaposition of the amino acids of the NLS is not sufficient forfunction, for example NLS function is generally not conferred by thepeptide having the same sequence of amino acids in the opposite order asthe NLS sequence. Adam, S. A. et al., 1989, Nature 337:276-79.

The primary structure, i.e., the linear sequence, of the NLS mostfrequently contains consecutive lysines, the N^(ε) moieties of whichpresumably closely approach one another, i.e., they are neighbors.However, certain functional NLS peptides lack consecutive lysines.Robbins, J., et al., 1991, Cell 64:615-23. Presumably the secondary andtertiary structure of these so called “bipartite” NLS peptides givesrise to neighboring N^(ε) moieties, which may be important for theiractivity.

Docking and subsequent movement of proteins across the nuclear porecomplex require transport factors. Import of NLS-containing proteinsacross the nuclear pore complex is mediated by karyopherin αβheterodimers (also termed NLS receptor/importin) which bindNLS-containing proteins in the cytosol and target them to the nucleus(Gorlich, D., et al., 1995, Curr. Biol. 5:383-392; Radu, A., et al.,1995, Proc. Natl. Acad. Sci. 92:1765-1773). Karyopherin α binds the NLS(Adam and Gerace, 1991, Cell 66:837-847) whereas karyopherin β enhancesthe affinity of α for the NLS (Rexach and Blobel, 1995, Cell 83:683-692)and mediates docking of karyopherin-NLS protein complexes tonucleoporins (a collective term for nuclear pore complex proteins) thatcontain FXFG peptide repeats. The GTPase Ran and its interacting proteinp10 (also termed NTF2) (Moore and Blobel, 1994, Proc. Natl. Acad. Sci.91:10212-10216) impart mobility to the translocation process bycatalyzing the disruption of karyopherin αβ heterodimers that havedocked to a nucleoporin (Nerhbass and Blobel, 1996, Science272:120-122). Partial reactions of the nuclear import can be reproducedin vitro using solution binding assays and recombinant karyopherins(Rexach and Blobel, 1995, supra).

Two inhibitors of the nuclear localization process have been described.Nuclear localization has been inhibited by lectins (e.g., wheat germagglutinin (WGA)) that bind to the O-linked glycoproteins associatedwith nuclear localization. Dabauvalle, M.-C., 1988, Exp.Cell Res.174:291-96; Sterne-Marr R., et al., 1992, J.Cell Biol. 116:271. Thenuclear localization process, which also depends upon the hydrolysis ofGTP, is blocked by a non-hydrolyzable analog of GTP, e.g., (γ-S)GTP,Melchior, F., 1993, J.Cell Biol. 123:1649.

However, neither (γ-S)GTP nor WGA can be used as pharmaceuticals.Proteins, such as WGA, can be introduced into the interior of a cellonly with considerable difficulty. The same limitation applies tothiotriphospates such as [γ-S]GTP. Further, GTPases are involved in amultitude of cell processes and intercellular signaling, thus, the useof a general inhibitor of GTPases would likely lead to unacceptable sideeffects.

2.3 The Significance of Nuclear Importation in HIV-1 Infections

Although HIV-1 is a retrovirus, it and other lentiviruses must bedistinguished from viruses of the onco-retrovirus group, which are notassociated with progressive fatal infection. For example, lentivirusesreplicate in non-proliferating cells, e.g., terminally differentiatedmacrophages, Weinberg, J. B., 1991, J.Exp. Med. 172:1477-82, whileonco-retroviruses, do not. Humphries, E. H., & Temin, H. M., 1974,J.Virol. 14:531-46. Secondly, lentiviruses are able to maintainthemselves in a non-integrated, extrachromosomal form in restingT-cells. Stevenson, M., et al., 1990, EMBO J. 9:1551-60; Bukrinsky, M.I., et al., 1991, Science 254:423; Zack, J. L., et al., 1992, J.Virol.66:1717-25. However, it is unclear whether this phenomenon is related tothe presence of latently infected peripheral blood lymphocytes (PBL) inHIV-1 infected subjects, wherein the virus is present in a provirusform. Schnittman, S. M., 1989, Science 245:305; Brinchmann, J. E., etal., 1991, J.Virol. 65:2019; Chapel, A., et al., 1992 J. Virol. 66:3966.

The productive infection of a cell by a retroviruses involves the stepsof penetration into the cell, synthesis of a DNA genome from the RNAgenetic material in the virion and insertion of the DNA genome into achromosome of the host, thereby forming a provirus. Both lenti- andoncoretroviruses gain access to the host cell's nucleus during mitosiswhen the nuclear membrane dissolves. However, the lentiviruses are alsoable to cross the nuclear membrane because viral proteins containingnuclear localization sequences are associated with the viralnucleoprotein complex.

The productive infection of terminally differentiated macrophageslocated in the central nervous system is thought to be responsible forthe dementia associated with AIDS. Keonig, S., et al., 1986, Science233:1089; Wiley, C. A. et al., 1986, Proc. Natl. Acad. Sci. 83:7089-93;Price, R. W., et al., 1988, Science 239:586-92. The infection ofterminally differentiated macrophages in the lymphoid system is known tocause aberrant cytokine production. Guilian, D., et al., 1990, Science250:1593; Fauci, A. S., et al., 1991, Ann. Int. Med. 114:678. Thus, thewasting syndrome associated with HIV-1, also known as “slim” disease, isbelieved to be a pathological process that is independent of the loss ofCD4-T-cells. Rather the pathobiology of the wasting is closely relatedto the pathobiology of cachexia in chronic inflammatory and malignantdiseases. Weiss, R. A., 1993, Science 260:1273. For these reasons, theinhibition on HIV-1 infection of macrophages and other non-dividingcells is understood to represent a highly desired modality in thetreatment of HIV-1 infection, especially for patients wherein dementiaor cachexia dominate the clinical picture.

Macrophages play an important role in the transmission of HIV as well.During early stages of the infection, macrophages and cells of themacrophage lineage (i.e. dendritic cells) may be the primary reservoirof HIV-1 in the body, supporting infection of T cells by antigenpresentation activities, Pantaleo, G., et al., 1993, Nature 362:355-358,as well as via the release of free virus. Direct cell-to-celltransmission of the virus may constitute the major route by whichinfection spreads during the early stages of the disease, afterresolution of the initial viremia.

It is noteworthy, in this regard, that macrophage-tropic strains ofHIV-1 predominate in the early stages of infection. Thus, it appearsthat the infection of macrophages is particularly important during thedevelopment of a chronic infective state of the host in a newly infectedsubject. Secondly, macrophages are the HIV-susceptible cell type mostreadily passed during sexual intercourse from an HIV-infected individualinto the circulation of an uninfected individual.

Finally, infection of quiescent T cells by HIV-1 has been shown to takeplace in vitro, Stevenson, M., et al., 1990, EMBO J. 9:1551-1560; Zack,J. A., 1990, Cell 61:213-222, and probably constitutes an importantpathway for the spread of infection in vivo at various stages of thedisease. Bukrinsky, M. I., et al., 1991, Science 254:423-427. AlthoughHIV-1 does not establish productive replication in quiescent T cells,the extrachromosomal retroviral DNA can persist in the cytoplasm of suchcells for a considerable period of time, and initiate replication uponactivation of the host cell. Stevenson, M., et al., 1990, EMBO J.9:1551-1560; Spina, C. A., et al., 1994, J. Exp. Med. 179:115-123;Miller, M. D., et al., 1994, J. Exp. Med. 179:101-113. A recent reportsuggests that the duration of viral persistence in the quiescent T celldepends on the presence of a functional NLS. von Schwedler, U., et al.,1994, Proc. Natl. Acad. Sci. 91:6992-6996. Thus, physicians recognizethe desirability of preventing the infection of macrophages by HIV andunderstand that substantial benefits would be obtained from the use of apharmacologic agent that prevents HIV infection in this cell type.

The mechanism whereby HIV, but not oncoretroviruses, infect non-dividingcells is now understood in broad outline. It is established that thefunction of the pre-integration complex of retrovirus in this regarddoes not depend upon the cellular mechanisms of mitosis or DNAreplication, per se. Rather the integration complex must merely gainaccess to nucleus. Brown, P. O., et al., 1987, Cell 49:347.Onco-retroviruses gain access to the nucleus upon the dissolution of thenuclear membrane in mitosis. By contrast, lentiviruses contain twodistinct proteins that mediate nuclear access through the nuclear porecomplex in the absence of cellular division. For the first of these, thematrix protein (MA or p17), nuclear importation activity is clearly dueto the presence of a trilysyl-containing NLS sequence. Bukrinsky, M. I.,et al., 1993, Nature 365:666; von Schwedler, U., et al., 1994, Proc.Natl. Acad. Sci. 91:6992. A second protein subserving the function ofnuclear entry, the vpr protein, does not contain an identifiable NLSconsensus sequence. Emerman, M., et al., 1994, Nature 369:108;Heinzinger, N. K. et al., 1994, Proc. Natl. Acad. Sci. 91:7311.

The significance of the NLS sequence in the importation of HIV-1 intothe nucleus of non-dividing cells has been illustrated in experimentswherein the presence in the medium of a high concentration (0.1 M) ofthe peptide having the sequence of the SV40 T-antigen NLS blocked theimportation of HIV-1 into the nucleus of aphidicolin-arrested CD4⁺ MT4cells. Gulizia, J., et al., 1994, J. Virol. 68:2021-25.

2.4 Infectious Diseases and Its Treatment

Treatment of an infectious disease with chemicals involves killing orinhibition of growth of the infectious agent, which may includefree-living and parasitic organisms. Parasitic diseases are widespreadin the animal world where a parasitic organism lives at the expense of ahost organism, and causes damage, or kills its host. Humans, domesticpets and livestocks are hosts to a variety of parasites. Parasites donot comprise a single taxonomic group, but are found within theprotozoans and metazoans, among other groups. In many ways, infectiousparasitic diseases resemble infectious diseases caused bymicrobiologicals such as fungi, bacteria and viruses.

Malaria remains one of the major health problems in the tropics. It isestimated that 300 million people a year are infected with malaria(World Health Organization, 1990, Malaria pp.15-27. In TropicalDiseases, Progress in Research 1989-1990, Geneva). Malaria istransmitted by Anopheles mosquitos in endemic areas, and often by bloodtransfusion in eradicated areas.

Malaria in humans is caused by at least four protozoan species ofPlasmodium: P. falciparum, P. vivax, P. ovale and P. malariae. Theasexual erythrocytic parasite, merozoite, is the stage in the life cyclethat causes the pathology of malaria with a characteristic pattern offever, chills and sweats. Anemia, acute renal failure and disturbancesin consciousness are often associated with malarial infection. P.falciparum can produce a large number of parasites in blood rapidly, andcauses the most morbidity and mortality.

The most important treatment of malaria to date is chemotherapy using anumber of natural and synthetic drugs. Antifolates, such aspyrimethamine, inhibit the parasite's dihydrofolate reductase, whereasthe aminoquinolines, such as chloroquine (4-aminoquinoline) have thedigestive vacuoles as their major site of action. Prior to theintroduction of chloroquine in the 1940's, quinine was the onlyeffective drug for treatment of malaria. Chloroquine is commonly used totreat acute infections with all four species, but has no effect onrelapses of infection by P. vivax or P. ovale. Chloroquine (500 mgweekly) may also be used to prevent malaria by suppressing the stagesthat multiply in the erythrocytes and cause the symptoms.

However, the use of these drugs in certain areas and in the future willbe seriously hampered by the emergence of drug resistant parasites.Chloroquine resistance is widespread and will continue to appear in newareas. Due to the possibility of resistance, the presence of parasitesin blood (i.e., parasitemia) is followed closely during treatment, andalternative drugs instituted if indicated. The decision on drug regimenwill depend on the origin of the infection. Combination therapy, such asquinine and Fansidar (pyrimethamine and sulfadoxine), is applied totreat chloroquine-resistant P. falciparum. Because of the presence ofmultidrug resistant P. falciparum in many parts of the world, preventionof malaria by chemoprophylaxis with currently available drugs is notalways effective.

In the last 20 years, only several drugs, such as mefloquine,halofantrine and artemisinin derivatives, have been developed to treatP. falciparum (Nosten et al., 1995, Drug Saf. 12:264-73). In view of thecontinuing spread of multidrug resistant P. falciparum, it is apparentthat novel effective chemotherapeutic agents are needed for use againstmalaria.

3 SUMMARY OF THE INVENTION

The present invention encompasses a class of alkyl aryl carbonylcompounds that forms stable binding interactions, preferably throughformation of reversible covalent bonds, with one or more basic aminoacid residues, wherein such basic amino acid residues are a part of anuclear localization signal (NLS). The stable binding interactionresults in the inhibition or neutralization of the nuclear localizationactivity of the NLS. The binding interaction is mediated by onefunctional component of the compound, i.e., the reactive group, whereasanother functional component of the compound, i.e. the targetting group,determines the specificity of the compound for different NLS.

This targetting function occurs by interaction of the targetting groupwith a docking site that is positioned proximately to the susceptiblebasic residues of the target NLS, such that docking of the compoundplaces it in a favorable configuration to form a stable interaction withbasic amino acid residues of the target NLS. The docking site is locatedeither on the same NLS-bearing protein, or on another component of alarger molecular complex that includes the NLS-bearing protein.

Preferred compounds of the invention provide divalent aryl carbonylmoieties as the reactive group, particularly aryl bis(ketone), arylbis(α-diketone) or aryl bis(β-diketone), linked to a targetting group,preferably to a nitrogen-containing heterocyclic functionality via anN-linkage. Particularly preferred compounds provided are bis acetyl,propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked to apyrimidinium, pyrimidine or triazine moiety.

The invention further encompasses methods of using the compounds of theinvention to form tandem binding interactions with proteins havingneighboring basic residues. As used, herein, neighboring basic residuesare two basic amino acid residues of a protein, particularly lysine andarginine residues, the side chain amino or guanidino functions of whichapproach each other as closely as the bis carbonyl functions of thearylene bis(carbonyl) compounds of the invention, when the protein is inits natured conformation. As used herein neighboring, adjacent andjuxtaposed are equivalent terms in reference to amino or guanidinomoieties, and refer to the physical locations of the amino or guanidinomoieties in the structure of the native protein and not to the positionsof the basic amino acid residues themselves in the linear sequence.

In one embodiment of the invention, the compounds of the invention canbe used to react with neighboring nitrogenous moieties in lysine andarginine residues of nuclear localization sequence (NLS) of a protein,thereby inactivating the NLS. The invention also encompasses uses ofcompounds of the invention for specifically inhibiting importation of aNLS-containing protein or molecular complex comprising a NLS-containingprotein, into the nucleus of a cell. The carbonyl group(s) of thecompounds of the invention inactivates the NLS of a protein which isessential for nuclear translocation of the complex. The compounds of theinvention are targeted to the complex by the specific interactionbetween the targetting group, preferrably a nitrogen-containingheterocyclic targetting group, and a docking site on a molecule in thecomplex that is distinct from the NLS. The invention further encompassesmethods of screening for alkyl aryl bis(carbonyl) and bis(dicarbonyl)compounds that are capable of inhibiting importation of a specificNLS-containing protein or molecular complex comprising a NLS-containingprotein into the nucleus of a cell. The screening assays of the presentinvention allow a compound of the present invention to be identified andselected without advance knowledge of the specific docking site thatconfers specificity on the NLS inhibitory activity of the compound.

The invention further encompasses methods of inhibiting productiveinfection by HIV-1 of terminally differentiated (non-dividing cells),particularly macrophages, by inhibition of the importation of thecytoplasmic HIV-1 complex into the nucleus of cell. Particularly theinvention concerns the administration of the compound effective to blocksuch importation to a cell. Thus, in one embodiment, the inventionencompasses methods of using the above-described compounds to preventproductive infection of terminally differentiated macrophages andresting T-cells in HIV-1 infected subjects.

Without limitation as to theory, the compounds of the invention isbelieved to block HIV-1 replication by binding to reverse transcriptaseand formation of tandem Schiff bases with neighboring N^(ε) moieties oflysines in the nuclear localization signal of HIV matrix antigen. As aresult, the matrix antigen is unable to interact with karyopherin α ofthe host cell and the viral nucleoprotein complex does not pass acrossthe nuclear membrane via interaction with the nuclear pore transportcomplex and/or other cellular components.

Moreover, compounds of the present invention are also useful forinhibiting viral infection or nuclear translocation of viral proteins inproliferating cell populations, to the extent that such occurs in someof the cells in the population during periods of the cell cycle in whichthe nuclear membrane is intact. Such infection or nuclear translocationof proteins in a proliferating population of cells is susceptible totreatment with the compounds of the present invention on the same basisas non-dividing or quiescent populations would be susceptible.

The invention further encompasses methods of using the compounds of theinvention in treating or preventing infectious diseases such as thosecaused by parasites, particularly Plasmodium species that cause malaria.

4 BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C. The structures of exemplary Compounds No. 2, 11 and 13 are,respectively, FIGS. 1A, 1B, 1C.

FIGS. 2A-C. The effect of various concentrations of Compound No. 2 on RTactivity in the supernatant of HIV-1-infected monocytes. FIG. 2A:Multiplicity of Infection (MOI) 1 ng p24/10⁶ monocytes, cultured inpresence of M-CSF. FIG. 2B: MOI 8 ng p24/10⁶ monocytes, cultured inabsence of M-CSF. FIG. 2C: MOI 0.8 ng p24/10⁶ monocytes, cultured inabsence of M-CSF.

FIG. 3. The effect of various concentrations of Compound No. 2 on RTactivity in the supernatant of HIV-1-infected mitogen-stimulatedperipheral blood leukocytes at infected at 10 and 1.0 ng p24/10⁶ cells,FIGS. 3A and 3B, respectively.

FIGS. 4A-F. The structures of the compounds used in Example 7 are shownrespectively in FIGS. 4A-4F. FIG. 4A:2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride(CNI-0294). FIG. 4B:2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine (CNI-1194). FIG.4C: 2-amino-4-(3-acetylphenyl)amino-6-methylpyrimidine (CNI-1594). FIG.4D: 2-amino-4-(4-acetylphenyl)amino-6-methylpyrimidine (CNI-1794).Figure E: 3.5-diacetylaniline (CNI-1894). FIG. 4F:4-phenylamino-2-amino-6-methylpyrimidine (CNI-4594).

FIG. 5. Representative plasma concentrations over time in mice treatedwith CNI-1194. Female ND4 Swiss-Webster mice were given a single 50mg/kg injection intraperitoneally (circles) or orally (squares). Thecalculated plasma concentrations, in μg/ml, was then plotted against thetime of sampling.

FIGS. 6A-6B. Chromatograms of plasma extracts from animals treated withCNI-0294 or CNI-1594. Female ND4 Swiss-Webster mice were given a singlei.p. injection of 50 mg/kg CNI-0294 (A) or 20 mg/kg CNI-1594 (B). Thechromatogram shown for CNI-0294 was from the 2 hr time point, and thatfor CNI-1594 for the 1 hr time point. The peaks labeled “2” and “15” arethe parent peaks for CNI-0294 and CNI-1594 respectively. The other peaksin the chromatogram represent possible metabolites (labeled “x”) andendogenous plasma peaks.

FIGS. 7A-7D. The in vitro metabolism of the CNI compounds. The drugswere incubated with mouse liver post-mitochondrial supernatants andNADPH for various lengths of time. The chromatograms shown are from the60 min time point for (A) CNI-0294, (B) CNI-1194, (C) CNI-1594, and (D)CNI-1894. The peaks labeled “2, 11, 15, 18” refer to the parent compoundpeaks, and those labeled “a-n” to putative metabolite peaks thatincreased over time and were not present in control incubations. Alloff-scale peaks were single peaks, and the scale was chosen to allowpresentation of trace metabolite peaks.

FIGS. 8A-8D. The in vivo metabolism of the CNI compounds. Female ND4Swiss Webster mice received a single intraperitoneal dose of (A) 50mg/kg CNI-0294, (B) 50 mg/kg CNI-1194, (C) 20 mg/kg CNI-1594, or (D) 50mg/kg CNI-1894. In all four graphs, the open bar represents the peakarea of the parent compound and the black bars the apparent metabolitepeaks. The metabolite peaks shown are (from left to right in eachgraph): (a) peak “d” (see FIG. 7 for letter-designated peaks), peak “a”,peak “c”, and a peak eluting at 13 minutes; (b) peak “h”, peak “e”, peak“f”, peak “g”, a peak eluting at 14 minutes, and a peak eluting at 23minutes; (C) peak “j”, peak “i”, peak “l”, and a peak eluting at 14minutes; (D) peak “m”, peak “n”, and a peak eluting at 11 minutes. Thepeak area units are arbitrary and calculated by the HPLC operatingsystem.

FIG. 9. The activity of CNI-0294 against Plasmodium berghei infectedmice. Female ND4 Swiss Webster mice were infected with infectederythrocytes and then treated once daily, for four days, with 50 mg/kgCNI-0294, or with distilled water. Six hours after the last dose, thinblood smears were made from each of the animals and the parasitemia wasdetermined. The bars represent the median parasitemia (n=4 for controlsand n=5 for treated).

FIGS. 10A-10C. Binding of HIV-1 nucleoprotein complexes to karyopherinα.

A. Binding of HIV-1 to karyopherin α is mediated by both MA and Vpr.

Cytoplasmic extracts prepared 4 hours after infection of H9 cells withequivalent amounts (100 ng of p24 per 10⁶ cells) of wild-typeHIV-1_(NLHX) or variants carrying inactivating mutations in MA NLSor/and Vpr were divided in two aliquots. DNA was extracted from onealiquot and quantified by PCR using primers specific for the HIV-1 polgene (bottom panel). The obtained signal represented the total amount ofthe HIV-1 DNA in the cytoplasm. The second aliquot was incubated withGST-karyopherin α immobilized on Sepharose beads. HIV-1 DNA wasextracted from the beads and analyzed by PCR using pol-specific primers.The obtained signal represented the amount of HIV-1 pre-integrationcomplexes bound to karyopherin α.

B. Binding of HIV-1 pseudovirion nucleoprotein complexes to karyopherinα is mediated by MA NLS.

H9 cells were inoculated with HIV-like gag-env pseudovirions thatcontain gag RNA; equal amounts of wild-type (wt) and mutantpseudovirions that carry amino acid substitutions in the MA NLS (ΔMANLS) were used. Cytoplasmic extracts prepared from infected cells wereincubated with polyclonal anti-MA serum (ΔMA, lane 2), pre-immune serum(NSS, lane 3), or nothing (lanes 1, 4, and 5). Samples were then mixedwith GST-karyopherin α immobilized on glutathione Sepharose beads for 30min at 25° C. Nucleic acids were extracted from Sepharose beads andquantified by RT-PCR using primers specific for HIV-1 gag gene. Tocontrol for possible differences in cell entry of wild-type vs. mutantagents, HIV-specific nucleic acids were extracted directly fromcytoplasmic extracts and assayed by RT-PCR (lanes 4 and 5).

C. CNI-H0294 inhibits interactions of karyopherin α with HIV-1pre-integration complexes, but not with pseudovirion-derivednucleoprotein complexes.

Cytoplasmic lysates of H9 cells infected with HIV-1RF (upper panel) orHIV-like pseudovirions (bottom panel) were treated for 2 hours withvarious concentrations of CNI-H0294, and were then mixed withGST-karyopherin α immobilized on glutathione Sepharose beads. HIV-1 DNAor RNA that co-precipitated with karyopherin α was quantified as in Aand B.

FIG. 11. CNI-H0294 binds to recombinant RT in solution.

Twenty nmol of [¹⁴C]-CNI-H0294 were mixed with 0.28 nmol of recombinantMA or RT (a p51/p66 heterodimer) in 40 μl of binding buffer. Sampleswere incubated for 2 h at 37° C. in the presence or absence of 200 nmolof unlabeled CNI-H0294. MA and RT proteins were immunoprecipitated usingprotein G agarose and sheep polyclonal anti-MA (αMA) or rabbit anti-RT(αRT) sera, respectively. Pre-immune sera (PI) was used as control.Bound material was eluted from protein G using 0.1 M glycine buffer [pH2.8] and the radioactivity in the eluate was quantified in ascintillation counter.

FIGS. 12A-12C. CNI-H0294 interacts with RT to produce the anti-HIVeffect.

A. CNI-H3094 competes with CNI-H0294 for binding to RT.

Recombinant RT (0.2 μM) was incubated with 3.3 μM of [¹⁴C]-labeledCNI-H0294 and increasing concentrations of unlabeled (cold) CNI-3094.The amount of [¹⁴C]-CNI-H0294 that bound to RT was measured as in FIG.11.

B. CNI-H3094 reduces CNI-H0294-mediated inhibition of HIV-karyopherin αinteraction.

Cytoplasmic lysates prepared from H9 cells infected with HIV-1RF weretreated with 10 μM CNI-H3094 (lane 1) or with 1 μM CNI-H0294 and 10 μM(lane 5), 5 μM (lane 4), 1 μM (lane 3), or no CNI-H3094 (lane 2). Theamount of pre-integration complexes available for interaction withkaryopherin α was quantified as in FIG. 10.

C. CNI-H3094 inhibits anti-HIV activity of CNI-H0294 in monocytecultures.

Monocytes infected with HIV-1_(ADA) were cultured in the presence ofCNI-H0294 and CNI-H3094 in various concentrations. Nine days afterinfection, RT activity in culture supernatants was quantified. Theresults are presented as percent of total RT activity in untreatedcultures (control). Three independent samples were assayed for each drugconcentration, and the standard deviation was less than 15%.

FIGS. 13A-13B. Analysis of CNI-H0294 interactions with the HIV-1pre-integration complex.

A. CNI-H0294 does not disrupt the interaction between MA and HIV-1 cDNA.

Cytoplasmic extracts prepared from H9 cells infected with HIV-1RF weretreated with 10 μM CNI-H0294 (lanes 2, 3, and 4) or left untreated (lane1). Samples were divided into two aliquots. One aliquot was mixed withGST-karyopherin α immobilized on Sepharose beads and the HIV-1 DNA thatbound was quantified by PCR. The second aliquot was immunoprecipitated(IP) with anti-MA serum (αMA, lane 3) or with pre-immune serum (NSS,lane 4) as control.

B. CNI-H0294 reacts with MA, but only when MA is associated with theHIV-1 pre-integration complex.

Cytoplasmic extracts from HIV-1RF-infected H9 cells were treated with[¹⁴C]CNI-H0294 (10 μM). After borohydride reduction the extracts wereimmunoprecipitated with anti-MA (αMA), anti-IN (αIN), or pre-immuneserum (PI). As control, similar reactions were performed using lysatesof pseudovirion-infected cells which lack RT and thus do not bindCNI-H0294. Immunoprecipitated radioactivity was quantified in ascintillation counter.

FIGS. 14A-14B. CNI-H0294 inhibits MA-, but not Vpr-mediated binding ofHIV-1 pre-integration complexes to karyopherin α.

H9 cells were infected with equal amounts of wild-type HIV-1_(NLHX) orwith mutant HIV-1_(NLHX) that lack Vpr (Vpr⁻) or carry a mutation thatinactivates the MA NLS (MA NLS⁻). Infected cells were washed andincubated for 4 h. An aliquot of each sample was used to quantify thetotal HIV-1 DNA (FIG. 14A) and the rest was used to prepare cytoplasmicextracts. Extracts were incubated with 1 μM CNI-H0294 (FIG. 14B, lanes2, 4, 6, 8) or were left untreated (lanes 1, 3, 5, 7). The amount ofpre-integration complexes available for binding to karyopherin α wasdetermined as in FIG. 10.

FIG. 15. Proposed mechanism of CNI-H0294 action.

CNI-H0294 binds to HIV-1 pre-integration complexes via RT, and thenreacts with an adjacent MA NLS. This interaction prevents binding ofkaryopherin α to MA NLS; this nearly abolishes nuclear import of thepre-integration complexes. Note that Vpr binds to karyopherin α even inthe presence of CNI-H0294; this explains the low level of importdetected in the presence of the drug.

5 DETAILED DESCRIPTION OF THE INVENTION 5.1 The Compounds and Methods ofTheir Synthesis

The present invention encompasses a class of alkyl aryl carbonylcompounds that forms stable, but preferably reversible, and mostpreferably reversible covalent interactions with one or more basic aminoacid residues, wherein such basic amino acid residues are a part of anuclear localization signal (NLS). The stable covalent interactionresults in the inhibition or neutralization of the nuclear localizationactivity of the NLS.

Two structural features are involved in conferring such capabilities ofa compound of the invention: (a) a moiety comprising at least onecarbonyl group that reacts with the side chains of basic amino acidresidues in the target protein, i.e., the reactive group; and (b) asecond moiety, i.e., the targetting group, that interacts with aspecific docking site and determines the specificity of the compound fordifferent NLS.

Although the reactive group(s) of the compounds of the invention canreact reversibly with any susceptible basic side chains in a protein,e.g., arginines and lysines, the interaction between the targettinggroup and the docking site confers specificity to the activity of thecarbonyl group(s), such that the carbonyl group(s) react only withparticular target residue(s) in a protein. This targetting functionoccurs by interaction of the targetting group with a docking site thatis located proximately to the susceptible side chains of basic aminoacid residues of the target NLS, such that docking of the compoundplaces it in a favorable configuration to form a stable interaction withthe side chains of the basic amino acid residues of the target NLS. Itis to be understood that the docking site is located either on the sameNLS-bearing protein, or on another component of a larger molecularcomplex that includes the NLS-bearing protein.

Preferred compounds of the invention provide divalent aryl carbonylmoieties as the reactive group, particularly aryl bis(ketone) or arylbis(α-diketone), aryl bis(β-diketone), linked to a targetting group,preferably to a nitrogen-containing heterocyclic functionality via anN-linkage. Particularly preferred compounds provided are bis acetyl,propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked to apyrimidinium, pyrimidine or triazine moiety.

The compounds of the present invention form reversible adducts with atarget NLS containing protein. In particular, the compounds form Schiffbases with adjacent lysine residues, and other reversible adducts withadjacent arginine residues. Thus, the compounds of the invention, e.g.,aryl bis(ketone), can advantageously be used for inactivation of an NLSwhere the NLS comprises lysine residues. On the other hand, where theNLS contains arginine residues, aryl bis(diketone) compounds,particularly dimethyl-substituted compounds, i.e., those lacking amethylene hydrogen between the ketones, can be used advantageously.

In one particular embodiment, the compounds of the invention are capableof forming Schiff bases with lysine residues of a target NLS containingprotein in a molecular complex, and interacting with a specific dockingsite on a molecule in the molecular complex, said docking site beingpositioned proximately to the lysine residues in the protein. Because ofthe proximity of the target lysine residues to the docking site, theinteraction of the targetting group with the docking site localizes thecompound of the invention to the vicinity of the target lysine residues,thereby facilitating the reaction of the carbonyl group(s) of thecompound with the target N^(ε) group of lysine residue(s) in the NLS,and resulting in the formation of stable Schiff bases and inactivationof the NLS.

Specific compounds of the invention, e.g., compound No. 2 or CNI-H0294,or 2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium salts,and their synthesis are described in section 6 et seq, and are disclosedin earlier U.S. Pat. Nos. 5,574,040; 5,733,932; 5,703,086 and 5,840,893,and in earlier-filed U.S. patent application Ser. No. 08/470,103 each ofwhich is hereby incorporated by reference.

According to the invention, the compounds of the invention are alkylaryl carbonyl compounds of formula (I):

wherein A, independently, =CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃, C(CH₃)₂COCH₂CH₃ or the like to yield anacetyl, propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl,3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or3-oxo-2,2-dimethylpentanoyl substituted aniline; P=1 or 2; L is a linkergroup containing an S, O, N or C atom, e.g., —SO₂—, —O—, —NH—, —N=,—CH₂— or —CH=; K is 0 or a positive integer, preferably K=1; and whereinJ represents (i) a saturated or unsaturated, substitued orunsubstituted, straight or branched acyclic hydrocarbon group; (ii) asaturated or unsaturated, substitued or unsubstituted, straight orbranched acyclic group containing hetero atoms such as nitrogen, sulfuror oxygen; (iii) a substituted or unsubstituted, saturated or aromatic,mono- or poly-cyclic group having 3 to 20 carbon atoms; or (iv) asubstituted or unsubstituted, saturated or aromatic, mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen, sulfur or oxygen. The compounds of the invention can containone or more linker groups (L), however, if J contains a linker group asdefined above, K can be 0.

The acyclic and cyclic groups defined above may be saturated orunsaturated and may, if desired, bear one or more substituents.Exemplary of such substituents are alkyl, alkoxy, phenoxy, alkenyl,alkynyl, phenylalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl,alkyloxy, alkylthio, alkenylthio, phenylalkylthio, hydroxyalkyl-thio,alkylthiocarbamylthio, phenyl, cyclohexyl, pyridyl, piperidinyl,alkylamino, amino, nitro, mercapto, cyano, hydroxyl or a halogen atom.

For example, J can be a substituted or unsubstituted five or sixmembered ring having 1-4 hetero ring atoms, at least one of which isnitrogen, the remainder of which are selected from nitrogen, oxygen orsulfur, e.g., pyridine, pyrrole, imidazole, thiazole, isothiazole,isoxazole, furazan, pyrrolidine, piperidine, imidazolidine, piperazine,oxazole, tetrazole, pyrazole, triazole, oxadiazole, thiodiazole.Alternatively, J can be a substituted or unsubstituted polycyclic grouphaving 1 to 4 hetero ring atoms, one of which is nitrogen and theremainder of which are nitrogen, oxygen or sulfur, e.g., indole,quinoxaline, quinazoline, quinoline, isoquinoline, purine.

By the term “alkyl” as used herein is meant a straight or branched chainsaturated hydrocarbon group having from 1 to 20 carbons such as methyl,ethyl, isopropyl, n-butyl, s-butyl, t-butyl, n-amyl, isoamyl, n-hexyl,n-octyl and n-decyl. The terms “alkenyl” and “alkynyl” are used to meanstraight or branched chain hydrocarbon groups having from 2 to 10carbons and unsaturated by a double or triple bond respectively, such asvinyl, allyl, propargyl, 1-methylvinyl, but-1-enyl, but-2-enyl,but-2-ynyl, 1 methylbut-2-enyl, pent-1-enyl, pent-3-enyl,3-methylbut-1-ynyl, 1,1-dimethylallyl, hex-2-enyl and1-methyl-1-ethylallyl. The term “phenylalkyl” means the aforementionedalkyl groups substituted by a phenyl group such as benzyl, phenethyl,phenopropyl, 1-benzylethyl, phenobutyl and 2-benzylpropyl. The term“aryl” as used herein is meant to include a monocyclic, bicyclic,tricyclic or other polycyclic compounds, wherein at least one ring isaromatic including aromatic hydrocarbons or hetero-aromatic hydrocarbonshaving heteroaromatic atoms such as nitrogen, sulfur and oxygen. Theterm “hydroxy-alkyl” means the aforementioned alkyl groups substitutedby a single hydroxyl group such as 2-hydroxyethyl, 2-hydroxypropyl,3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl and 6-hydroxyhexyl. Theterms “alkylthio, alkenylthio, alkynylthio, hydroxy-alkylthio andphenyl-alkylthio” as used herein mean the aforementioned alkyl, alkenyl,alkynyl, hydroxy-alkyl and phenyl-alkyl groups is linked through asulfur atom.

The term “substituted” as used herein means that the group in question,e.g., alkyl group, aryl group, etc., may bear one or more substituentsincluding but not limited to halogen, hydroxy, cyano amino, nitro,mercapto, carboxy and other substituents known to those skilled in theart.

The terms “saturated” as used herein means an organic compound withneither double or triple bonds. The term “unsaturated” as used hereinmeans an organic compound containing either double or triple bonds.

In another embodiment, the compounds of the invention are formedaccording to formula (II):

wherein A, independently, =CH₃ or CH₂CH₃ and P=1 or 2; and

wherein X=NH₂, CH₃ or CH₂CH₃; X′=CH₃ or CH₂CH₃; Y=NH₂, NHCH₃, N(CH₃)₂,1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently, =H, NH₂, NHCH₃, N(CH₃)₂ or N⁺(CH₃)₃,1-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.

In a preferred embodiment the compounds of the invention are bis ketonearylene compounds having a third nitrogenous substituent. Thenitrogenous substituent can be further substituted with an aromaticnitrogen-containing heterocyclic compound.

More precisely the bis ketone arylene compounds of the invention areformed according to the formula (III):

wherein A=CH₃ or CH₂CH₃ and

wherein X=NH₂, CH₃ or CH₂CH₃; X′=CH₃ or CH₂CH₃; Y=NH₂, NHCH₃, N(CH₃)₂,1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently, =H, NH₂, NHCH₃, N(CH₃)₂ or N⁺(CH₃)₃,1-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.

In yet another embodiment of the invention, the compounds of theinvention are compounds formed according to formula (I), wherein J isnot R; L is not —NH—; and wherein

and wherein, independently, X=NH₂, CH₃ or CH₂CH₃; X′=CH₃ or CH₂CH₃;Y=NH₂, NHCH₃, N(CH₃)₂; and Z=H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently, =H, NH₂, NHCH₃, N(CH₃)₂ or N⁺(CH₃)₃;and salts thereof.

The compounds of the present invention can be synthesized by reactinganiline—to form a compound of formula (II), wherein P is 0—or an acetylor propanoyl derivative of aniline—to form a compound of formula (II),wherein P is 1—or a diacetyl or dipropanoyl derivative of aniline—toform a compound of formula (II) or formula (III) wherein P is 2—with achloro derivative of purine, aminomethylpyrimidine, diamino-triazine, orwith a cyanoguanidine. The reaction can be performed at 90-100° C. in anaqueous solvent in the presence of a mineral acid to yield thecorresponding aminophenyl pyridine or triazine. The pyrimidinium can besynthesized from the pyrimidine by reaction with an excess methyl iodideat 40-45° C. under reflux conditions in 1:1 acetonitrile/tetrahydrofuranor in a 1:1:2 mixture of dichloromethane/acetonitrile/tetrahydrofuran.

For synthesis of aryl bis(diketone) compounds, the acyl groups attachedto the benzene ring, for example in 3,5-diacetylaniline, may beconverted to 2-oxoacyl groups by reaction with selenium dioxide orselenious acid in wet dioxane or other suitable medium (Rabjohn, N.,1976, Org. React. 24:261-415). Acyl groups attached to benzene rings maybe converted to higher-chain 3-oxoacyl groups by treatment with strongbase such as sodium hydride or sodium metal in the presence of analiphatic ester such as ethyl acetate, when the acyl groups are of thestructure X—CH₂—CO— (March, J., 1992, Advanced Organic Chemistry, 4thed., Wiley Interscience, New York, pp491-493; Fenton, D. E. et al.,1980, Inorg. Chim. Acta 44:L105-L106)

Any chemistries for generating chemical libraries known in the art canbe used to form the J group in the compound of formula (I), includingbut not limited to combinatorial chemistries, in which interchangeablechemical building blocks are systematically assembled to provide diversestructures. See generally Gordon, E. M. et al., 1994, J. Med. Chem.37:1385; Chen, C. et al., 1994, J. Amer. Chem. Soc. 116:2661; Cho, C. Y.et al., 1993, Science 261:1303. The modification and adaptation of thevarious chemistries for generating diversities for the formation of theJ group in the compound of formula (I) will be apparent to persons ofskill in the art. Moreover, automated synthesis systems can be used togenerate the desired chemical diversities including, for example,workstations and robots made by Takeda Chemical Industries Ltd, Osaka,Japan; Zymark Corporation, Hopkinton, Mass.; and Hewlett Packard, PaloAlto, Calif.

5.2 The Inhibition of HIV-1 Importation into the Nucleus of Non-DividingCells

A quantitative measurement of the activity of the compounds of theinvention to block the replication of HIV-1 in non-dividing cells can bedetermined by culture of a macrophage-tropic strain of HIV-1 onperipheral blood-derived macrophages. The cells are cultured for 5-6days prior to infection in a medium consisting of DMEM supplemented with10% type A/B human serum and 200 U/ml Macrophage Colony StimulatingFactor, with half the medium changed after 3 days, to reach a density ofabout 10⁶ cells per 5 ml well. A macrophage-tropic viral stock may begrown on these cells. The concentration of infectious particles in thestock is estimated by measurement of p24 antigen concentration.

To test the effect of compounds of the invention on HIV-1 infection inthe above-described culture system, the medium is removed and replacedwith medium containing HIV-1 at a concentration of 1 ng of p24 (10⁴TCID₅₀/ml (TCID=tissue culture infectious doses)) and a knownconcentration of the compound of the invention (the inhibitor). After 24hours, the cultures are washed to remove non-adherent virus and theculture is re-fed with medium containing the inhibitor at the desiredconcentration. The amount of replication of HIV-1 is estimated by anassay of the reverse transcriptase activity or by an assay of theconcentration of p24 antigen in the culture medium every 2-3 daysthroughout the post-infection period. In a preferred embodiment theanti-HIV potency of the candidate drug is measured by comparison of theconcentration of reverse transcriptase (RT) or of p24 antigen in themedium of the treated and control cultures at the time of the peak ofthese values in non-treated control cultures, that is about day 5 or 6post-infection. Repetition at various levels of inhibitor allows for thecalculation of the concentration of inhibitor that achieves 50%inhibition of viral growth, IC₅₀. Table I discloses the IC₅₀ of variousinhibitors.

TABLE I Compound IC₅₀ 2-amino-4-(3,5-diacetylphenyl)amino-1,6-  1 nMdimethylpyrimidinium iodide (Compound No. 2)2-amino-4-(3-acetylphenyl)amino-1,6- 10 nM dimethylpyrimidinium iodide(Compound No. 14) 2 amino-4-(3,5-diacetylphenyl)amino-6- 50 nMmethylpyrimidine (Compound No. 11) 4-(3-acetylphenyl)amino-2-amino-6- 15nM methylpyrimidine (Compound No. 15)

Alternatively, the compounds may all be compared for inhibition of HIVreplication at a fixed concentration. Presented in Table II arecompounds that were used at a concentration of 100 nM to inhibit theproduction of HIV-1 in cultured monocytes infected with HIV-1 10 daysprior to assay (10 ng of p24/10⁶ cells). The production of HIV-1 in eachtreated culture is reported as percentage of untreated control.

TABLE II Viral Compound Production N-(3,5-diacetylphenyl)biguanidehydrochloride 12% (Compound No. 12)2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine 14% (Compound No.13) 4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine 20% (Compound No.17) 3,5-diacetylaniline 20% N,N-dimethyl-3,5-diacetylaniline 25%2,6-diacetylaniline 28% 3,5-diacetylpyridine 58%

FIG. 2A presents further results of the use of the most active of thecompounds of Table I, Compound No. 2, to block the replication of HIV-1in purified monocytes, cultured in medium supplemented withmonocyte-colony stimulating factor (M-CSF). The cultures were treatedwith none or between 10⁻¹² and 10⁻⁶ M Compound No. 2 and, simultaneouslywith the beginning of treatment, the cells were exposed to themonocyte-tropic strain HIV-1_(ADA) at about 0.01 TCID₅₀/cell (1 ngp24/10⁶ cells) for 2 hours. Samples were withdrawn at days 3, 6, 10, 14and 17 after infection and assayed for reverse transcription activity.Compound No. 2 does not inhibit reverse transcriptase, data not shown.The results show that under these conditions the IC₅₀ concentrations isbetween 0.1 and 1.0 nM and that a concentration of between 0.1 μM and1.0 μM completely inhibits the replication of the virus.

FIGS. 2B and 2C show the effects of various concentrations of CompoundNo. 2 on the production of HIV-1 in monocyte cultures not supplementedwith M-CSF. In these studies MOI, as determined by concentration of p24antigen was; FIG. 2B (8 ng/10⁶ cells) and FIG. 2C (0.8 ng/10⁶ cells).These experiments showed IC₅₀s of about 10 nM and of less than 1.0 nMrespectively.

The inhibition of the replication of HIV-1 is not due to generalcytotoxic effects of the compound. Concentrations of Compound No. 2 ashigh as 10 μM were without toxic effects on the monocyte cultures asdetermined by lactate dehydrogenase release and trypan blue exclusion.Further evidence of the specificity of the inhibition due to CompoundNo. 2 is provided by the data presented in FIGS. 3A and 3B whereinmitogen-stimulated peripheral blood leukocytes were cultured inIL-2-supplemented medium and were exposed to the HIV-1_(ADA) at p24concentrations of 10 and 1 ng/10⁶ cells, respectively. In thisexperiment up to 10 μM Compound No. 2 had only a marginal effect onviral production at the higher MOI. At the lower MOI, 1 and 10 μM ofCompound No. 2 caused an approximate 2-fold reduction in viral output.

The inhibition of HIV-1 importation into the nucleus of non-dividingcells can also be directly measured. One suitable method to determinedirectly the activity of compounds of the invention utilizes a cell linethat is susceptible to HIV-1 infection, e.g., MT-4 cells, that is growtharrested by treatment with aphidicolin and exposed to HIV-1. PCRamplification is used to detect double-stranded closed circular HIV-1genomes, which are formed only after nuclear importation, by selectingprimers that bridge the junction point of the genome. For greater detailsee Bukrinsky, M. I., et al., 1992, Proc. Natl. Acad. Sci. 89:6580-84.

5.3 The Treatment of HIV Infection

The present invention provides a method of treatment of HIV-1 infectionby administering to an HIV-1-infected subject a pharmaceuticalcomposition having, as an active ingredient, an effective amount of acompound of formula (I) and (III), and particularly a compound offormula (III). In one embodiment the compound to be administered isCompound No. 2. Pharmaceutical compositions suitable for oral,intraperitoneal, and intravenous administration can be used in thepractice of the invention. Such pharmaceutical compositions include, byway of non-limiting examples, aqueous solutions of the chloride,bicarbonate, phosphate and acetate salts of Compound No. 2 andpH-buffered mixtures thereof. The chloride salt of compound 2 is hereinreferred to as CNI-0294. Compound 11, Compound 14 and Compound 15 arealso known as CNI-1194, CNI-H1494 and CNI-1594, respectively.

The effective dose of the active ingredient can be determined by methodswell known to those skilled in medicinal chemistry and pharmacology. Aneffective dose is the dose that achieves in the subject's plasma aconcentration of the active ingredient that is sufficient to inhibit thereplication of HIV-1 in monocyte cultures as described in Section 5.4,supra, but does not lead to cytopathic effects in such cultures.

The daily dose and dosing schedule to be given a subject can bedetermined by those skilled in the art, using the pharmacokineticconstants set forth in Table III below, to achieve a target plasmaconcentration. The target plasma concentration can be selected byroutine pharmacological and clinical investigation methods well-known tothose skilled in the art, and can be based on a range of concentrationswhich encompass the IC₅₀ calculated for each particular compound. Forexample, the dose can be adjusted to achieve a range of target plasmaconcentrations that included the IC₅₀ for the compounds as shown inTable I above.

TABLE III Pharmacokinetic parameters of the CNI compounds. CNI- CNI-CNI- CNI- CNI- CNI- CNI- 0294 0294 0294 1194 1194 1594 1894 Route ofi.p. i.p. oral i.p. oral i.p. i.p. Injection Dose (mf/kg) 50 50 50 50 5020 50 Vehicle DP* W* DP W W W W AUC (μ*hr/ml) 9.15 8.83 0.56 3.93 0.570.82 20.20 C_(max) (μg/ml) 18.76 18.93 0.41 5.70 0.35 1.93 13.43 t_(max)(min) 5 5 60 15 15 15 5 α (hr⁻¹) 1.12 1.74 — 1.83 — 2.14 1.19 β (hr⁻¹)0.15 0.19 — 0.19 — 0.04 0.03 A (μg/ml) 14.00 16.07 — 5.22 — 1.10 14.93 B(μg/ml) 0.07 0.05 — 0.14 — 0.01 0.15 t_(½μ)(hr) 0.62 0.40 — 0.38 — 0.320.58 t_(½β)(hr) 4.62 3.65 — 3.65 — 17.33 23.10 V_(D) (L) 14.14 19.80 —5.21 — 39.60 6.60 Cl_(tot) (ml/min) 35.35 62.70 — 16.50 — 26.40 3.30Bioavailability — — 0.06 — 0.15 — -- *DP = DMSO/peanut oil, W = water

For example, using the foregoing pharmacokinetic constants,particularly, the clearance rate, the daily dose and dosing scheduleneeded to obtain a given target average plasma concentration can becalculated. The results of such calculations for Compound Nos. 2, 11 and15 are presented in Table IV. The calculated doses of Compound Nos. 2and 15 are considerably below the toxic levels, as measured by the LD₅₀,of these compounds. See, Section 6.4 below.

TABLE IV Target Compound serum Clearance‡ Dose No. M.W. conc. (ml/min)(mg/Kg day)  2* 334 10 nM 35.35 6.80 11 280 50 nM 16.50 13.3 15 250 15nM 26.40 5.70 ‡measured in a 25 gr mouse *Chloride salt (CNI-0294)

Using such methods, a dose can be calculated to achieve a predeterminedtarget plasma concentration. A practicable target plasma concentrationof Compound No. 2 ranges from 0.5 nM to 10 nM; for Compound No. 11, apracticable target range is from 25 nM to 100 nM; for Compound No. 15, apracticable target range is from 7.5 nM to 50 nM.

Subjects who can benefit from the administration of the compounds of theinvention according to this method include all persons infected byHIV-1. More particularly, firstly, those who benefit include thosesubjects who have or are at risk to develop CNS signs of HIV-1 infectionand/or subjects that have developed significant weight loss. Secondly,those who benefit include those who have been recently exposed to HIV-1,but who do not yet have an established chronic infection.

5.4 Pharmaceutical Formulations

Because of their pharmacological properties, the compounds of thepresent invention can be used especially as agents to treat patientssuffering from HIV and can be used as agents to treat patients sufferingfrom other viral infections or chronic diseases that are dependent uponnuclear localization as part of the pathogenic process. The compounds ofthe invention can also be used to treat or prevent other infectiousdiseases such as parasitic diseases, and in particular malaria. Such acompound can be administered to a patient either by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s).

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well-known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

5.5 Use of the Compounds of the Invention to Derivatize Proteins

The compounds of the present invention of formula (III), wherein P is 1or 2, can be used to derivatize a target protein and thereby determinethe presence of adjacent N^(ε)-moieties. The test reaction can beconducted in aqueous buffer at mild to moderate alkaline pH, betweenabout 7.2 and 8.0. Specific derivatization of the target protein can bedetected by any means that separates protein-bound and free derivatizingcompound. The derivatizing compound optionally can be detected byradiolabeling it. In one embodiment, the compound can be synthesizedusing ¹⁴C-methyliodide in place of methyliodide. Alternatively, use canbe made of the strong UV absorption or fluorescence of the derivatizingcompounds. Compound No. 2, for example has a absorption peak of 16,000M⁻¹ cm⁻¹ at λ=298 nm. In a preferred embodiment the target protein isderivatized by a compound of the invention, irreversibly reduced withsodium borohydride or cyanoborohydride and fragmented into peptides bytrypsin or the like. The resultant peptides can be compared with thepeptides obtained from an unreacted sample of the protein by analysisusing any chromatographic or electrophoretic technique that resolvespeptides, e.g., reverse phase High Performance Liquid Chromatography(HPLC). When the peptides are resolved by any high resolutionchromatography procedure, the derivatized peptides can be readilydetected by their altered elution time and the absorbance at λ=298 nm.

In a preferred embodiment the practitioner will conduct the reaction atvarious pH points to determine whether a positive result can be obtainedat any point within the expected range. A positive result, i.e., aresult that indicates the presence of adjacent N^(ε)-moieties, is one inwhich a large fraction of each of a limited number, i.e., between 1-4,of peptides of the target protein are derivatized and negligible amountsof other peptides are affected.

The above-described protein derivatization technique can be used todetermine whether a candidate compound can be used, according to theinvention to prevent productive HIV-1 infection of macrophages. Acomparison of the activity of a candidate compound and that of CompoundNo. 2 as derivatizing agents specific for nuclear localization sequencescan be made. A compound that derivatizes the same peptides to the sameextent as Compound No. 2 can be used to practice the invention.

5.6 The Treatment of Infectious Diseases

The compounds of the present invention can be used to prevent or treatinfectious diseases in animals, including mammals and preferably humans,and these compounds are particularly suited to treatment of parasiticdiseases, more particularly, malaria. The invention described hereinprovides methods for treatment of infection, including and withoutlimitation, infection with parasites, and methods of preventing diseasesassociated with such infection. The compounds can reduce parasitemiawhen administered to an animal infected with a parasite.

Infectious diseases may include without limitation: protozoal diseasessuch as those caused by Kinetoplastida such as Trypanosoma andLeishmania, by Diplomonadina such as Giardia, by Trichomonadida such asDientamoeba and Trichomonas, by Gymnamoebia such as Naegleria and theAmoebida such as Entamoeba and Acanthamoeba, by Sporozoasida such asBabesia and the Coccidiasina such as Isospora, Toxoplasma,Cryptosporidium, Eimeria, Thelleria, and Plasmodium; metazoal diseasessuch as those caused by the Nematoda (roundworms) such as Ascaris,Toxocara, the hookworms, Strongyloides, the whipworms, the pinworms,Dracunculus, Trichinella, and the filarial worms, and by thePlatyhelminthes (flatworms) such as the Trematoda such as Schistosoma,the blood flukes, liver flukes, intestinal flukes, and lung flukes, andthe Cestoda such as the tapeworms; viral and chlamydial diseasesincluding for instance those caused by the Poxviridae, Iridoviridae,Herpesviridae, Adenoviridae, Papovaviridae, Hepadnaviridae,Parvoviridae, Reoviridae, Birnaviridae, Togaviridae, Coronaviridae,Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae,Bunyaviridae, Arenaviridae, Retroviridae, Picornaviridae, Calciviridaeand by Chlamydia; bacterial diseases; mycobacterial diseases;spirochetal diseases; rickettsial diseases; and fungal diseases.

In one embodiment, the compounds of the invention having anti-infectiveactivity are formed according to formula (I), (II) and (III) asdescribed in section 5.1.

In another embodiment, the compounds of the invention may be usedtherapeutically against infections with Plasmodium species such as P.falciparum, P. vivax, P. ovale and P. malariae, that cause acute andrecurrent malaria in humans. The compounds of the invention are alsoactive against infection by other Plasmodium species, which include P.berghei, P. knowlesi, P. simium, P. cynomolgi bastianelli and P.brasilianum.

In yet another embodiment of the invention, the compounds may be usefulin providing chemoprophylaxis for individuals at risk of infection, suchas when travelling in endemic areas. By maintaining in circulation aneffective concentration of a compound of the invention, malaria can beprevented by suppressing the pathological stages of infection withPlasmodium species. Without being bound by any theory, the compounds ofthe invention can be effective against various stages of the life cycleof the parasite, including sporozoites and merozoites, as well asdormant, asexual and sexual stages. The compounds of the invention maybe active in the blood stream, in erythrocytes, in the liver, or inother tissues where the malaria parasite may reside.

In a specific embodiment of the invention, the compound of the inventioncan be used to prevent malaria, or to treat malaria, or to treatinfection with Plasmodium species that are resistant to antimalarialdrugs, such as, but not limited to, chloroquine and pyrimethamine. Theantimalarial properties of the compounds are not diminished against P.falciparum known to be resistant to chloroquine or pyrimethamine (seesection 8 infra). Although not wishing to be bound by any theory ofmechanism of the compounds, it is contemplated that the compoundsinteract with biochemical targets that are different and independentfrom those affected by these two classic antimalarial drugs. Thus, thecompounds of the invention may be used preferentially to treat malarialinfections arising out of areas that are known or suspected to harbordrug-resistant Plasmodium species.

In a further embodiment, the compounds may contain a single acyl group,i.e., P=1, on the arylene ring or the acyl group can be absenttherefrom, i.e., P=0, and/or the heterocyclic substituent, i.e., R, canbe uncharged. In the embodiment of the invention wherein there are twoacyl groups, i.e., P=2, on the arylene ring, it is preferred that suchacyl groups are not in an ortho arrangement relative to each other. Inanother preferred embodiment of the invention, the compounds thatpossess potent antimalarial activity are arylene bis(methylketone)compounds that contain a charged heterocylic ring such as apyrimidinium, as in CNI-0294 (see FIG. 4A).

The antimalarial properties of the compounds of the invention can beanalyzed by techniques, assays and experimental animal models well knownin the art. For example, the inhibition of growth of Plasmodiumfalciparum in vitro by the compounds may be assessed by thehypoxanthine-incorporation method (Desjardins et al., 1979, Antimicrob.Ag. Chemother. 16:710-718). The in vitro antiparasitic activities ofseveral exemplary compounds of the invention were assessed by thismethod, and the results are described in Section 8. The in vivo efficacyof the compounds can also be tested in mouse models in which parasitemiais enumerated following administration of the compound (Ager, A. L.1984, Rodent malaria models, pp 225-264. In Handbook of ExperimentalPharmacology vol. 68, Antimalarial Drugs, Peters and Richards eds,Springer-Verlag, Berlin). The in vivo activity of several exemplarycompounds have been evaluated in a four-day suppression model in mouse,and the results are provided in Section 8.

The present invention also provides pharmaceutical compositions. Suchpharmaceutical compositions comprises a prophylactically ortherapeutically effective amount of the compound and a pharmaceuticalcarrier, such as those described in section 5.4. More specifically, aneffective amount means an amount effective to prevent development of orto alleviate the existing symptoms of the subject being treated.Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. For any compound used in the method of the invention,the effective dose can be estimated initially from in vitro assays. Adose can be formulated in animal models to achieve a circulating rangethat includes the IC₅₀ (i.e., the concentration of compound whichachieves a half-maximal inhibition of growth of parasite) as determinedin the in vitro assay. Such information can be used to more accuratelydetermine useful doses in subjects, for example, humans. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration. Various delivery systems are known and can beused for administration of the compound, e.g., encapsulation inliposomes. Other methods of administration include but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal and oral routes.

In another embodiment, the invention provides a method of preventing ortreating malaria by administering to a subject in need thereof aneffective amount of a compound of the invention. In a further aspectthere is provided a method of preventing or treating malaria, especiallymalaria caused by drug resistant Plasmodium species in humans, whichmethod comprise administering to the individual in need thereof aneffective amount of a compound of the present invention and an effectiveamount of an antimalarial drug. The invention also provides the use of acompound of the invention and an antimalarial drug in the manufacture ofa medicament for the prevention or treatment of malaria. Suchantimalarial drugs may include but are not limited to quinine,aminoquinolines (chloroquine and primaquine), pyrimethamine, mefloquine,halofantrine, and artemisinins.

The “adjunct administration” of a compound of the invention and anantimalarial drug means that the two are administered either as amixture or sequentially. When administered sequentially, the compoundmay be administered before or after the antimalarial drug, so long asthe first administered agent is still providing antimalarial activity inthe animal when the second agent is administered. Any of theabove-described modes of administration may be used in combination todeliver the compound and the antimalarial drug.

The present invention is to be understood as embracing all such regimensand the term “adjunct administration” is to be interpreted accordingly.When a compound of the invention and an antimalarial drug areadministered adjunctively as a mixture, they are preferably given in theform of a pharmaceutical composition comprising both agents. Thus, in afurther embodiment of the invention, it is provided a pharmaceuticalcomposition comprising a compound of the invention and an antimalarialdrug, together with a pharmaceutically acceptable carrier.

5.7 Compounds and Assays for Compounds that Target Specific NLS

The present invention also provides methods of use of the alkyl arylcarbonyl compounds as described in section 5.1. The reactive group(s) ofthe compounds of the invention are capable of forming stable butreversible covalent interactions with the side chain of basic amino acidresidue(s) of a protein. In certain embodiments of the invention, thedivalency of the compound ensures that the compound will form especiallystable associations with sequences, such as nuclear localization signals(NLS), that comprise multiple basic amino acid residues, such as lysinesand arginines. Nuclear localization signals, in general, and the HIVmatrix antigen (MA) p17 NLS in particular are characterized by a stretchof basic, and often positively charged, amino acids typically includingone or more lysines. Such NLS-containing proteins are often associatedwith other molecules in a complex.

The interaction of basic side chain(s) with the reactive carbonylgroup(s) of the compound is facilitated by the prior interaction of thetargetting group of the compound with a specific docking site, saiddocking site being positioned proximately to the side chain of the basicamino acid residue(s) in the target NLS. The docking site may be locatedon the NLS-containing protein or another molecule in a complexcomprising the NLS-containing protein. As a result of the formation ofstable but reversible covalent interactions with the compound, theNLS-containing protein or the molecular complex comprisingNLS-containing protein is prevented from interacting with cellularreceptors for the NLS. The protein or molecular complex is thus blockedfrom importation into the nucleus.

Although the carbonyl group(s) of the compounds of the invention canreact with alternative susceptible side chains in a protein, theinteraction between the targetting group and the docking site confersspecificity to the activity of the carbonyl group(s), such that thecarbonyl group(s) react only with particular target basic amino acidresidues in a protein. The specific recognition and binding of thecompound to a docking site determines which basic amino acid residue(s)in a protein will become inactivated by the carbonyl group(s) of thecompound. Because of the proximity of the basic residues in the targetNLS to the docking site, the interaction of the compound with thedocking site localizes the compound to the vicinity of the target NLS,thereby facilitating the reaction of the carbonyl group(s) of thecompound with the target NLS. It is to be understood that the dockingsite can be on the NLS-containing protein or another molecule in acomplex comprising the NLS-containing protein. Thus, in one embodiment,the compounds of the invention can be used to target a specific nuclearlocalization signal, thereby blocking importation of specific proteinsor molecular complexes into the nucleus.

As demonstrated by the experiments in section 9, the compounds of theinvention, particularly Compound No. 2 or CNI-H0294, are capable ofinhibiting nuclear translocation of HIV-1 pre-integration complexes. Theinhibitory effect is caused by the inactivation of the NLS of HIV MA ina reaction that requires the presence of HIV reverse transcriptase (RT),i.e. the target nucleoprotein complex. The carbonyl groups of CompoundNo. 2 or CNI-H0294 react with the NLS of MA by formation of Schiff baseadducts, and prevent the binding of MA to karyopherin α, the cellularreceptor for NLS, and that blocks translocation of proteins into thenucleus. The inventors further showed that the interaction betweenCompound No. 2 or CNI-H0294 via its pyrimidine moiety with RT thatcontains a docking site determines the specificity of the compoundtowards HIV pre-integration complex, and its low cytotoxicity to thehost cell. Thus, the alkyl aryl carbonyl compounds of the invention canbe used for targeted inactivation of NLS in a protein or a molecularcomplex, such as the HIV-1 or other viral pre-integration complex,comprising contacting the protein or molecular complex with a compoundof the invention. Furthermore, variations and modifications of thetargetting group provides for altered binding specificities, and canserve to target the alkyl aryl carbonyl compound to a differentNLS-containing protein or molecular complex. Thus, it is envisioned thatthe alkyl aryl carbonyl compounds of the invention, can be selected toinactivate specifically various different NLS-containing proteins ormolecular complexes.

Accordingly, it is contemplated that a molecular complex comprises atleast one NLS-containing protein and can include other proteins, nucleicacids, carbohydrates, lipids and other biological molecules. The dockingsite to which the targeting group of the compound binds may reside onany molecule in the molecular complex. In a preferred embodiment, thetarget of the compound of the present invention is a viral nucleoproteincomplex or a viral protein. Alternatively, a target may be a cellularprotein such as a transcription factor. An exemplary, non-limiting listof such targets may include, viruses which translocate both viralproteins and nucleic acids into the infected cell nucleus, such as humanimmunodeficiency virus and other retroviruses, influenza virus,hepatitis B and hepatitis delta virus, papillomaviruses andparvoviruses; viruses which translocate only viral protein into theinfected cell nucleus such as adenoviruses, measles and otherparamyxoviruses, herpes viruses, and rabies and other bunyaviruses; andsingle protein, such as NF-κB and other transcription factors.

In another embodiment of the invention, screening assays are providedfor identification of alkyl aryl carbonyl compounds of the inventionthat can inactivate the NLS of a specific NLS-containing protein orNLS-containing molecular complex. The assays of the invention involvesmonitoring the binding of the NLS-containing protein or molecularcomplexes to at least one protein or fragment thereof that interactswith the NLS in the course of nuclear translocation, i.e., the cellularreceptor, in the presence of an alkyl aryl carbonyl test compound of thepresent invention, and a comparison of such binding in the absence ofthe test compound. It is anticipated that the desired compound bindsspecifically to a docking site in the NLS-containing protein ormolecular complex, and inactivates the NLS. As a result, the binding ofthe NLS protein or molecular complex to the cellular receptor is reducedor abolished.

Any cellular protein(s) that interact with the NLS, and functionallyeffect or contribute to the nuclear translocation of the NLS-containingprotein or complex can be used in the assays of the invention. Fragmentsof such cellular proteins that bind NLS can also be used in the assaysof the invention. Such cellular proteins and fragments thereof,collectively referred herein to as the cellular receptor moiety, mayinclude but are not limited to, karyopherin αβ heterodimer and fragmentsthereof, or fusion proteins, such as karyopherin α-glutathioneS-transferase (GST-karyopherin α).

The assays are performed in vitro in which the cellular receptor moietyis immobilized directly or indirectly onto a solid support. TheNLS-containing protein or molecular complex, purified or in a cellextract, is contacted with the immobilized cellular receptor moiety inthe presence of test compound. As a control, the NLS-containing proteinor molecular complex is contacted with the immobilized cellular receptormoiety under the same condition, but in the absence of the testcompound. After an interval sufficient for binding reactions to occuramong the components in the assay, the solid support is washed to removeany unbound molecules. A detection procedure is performed with the solidsupport to quantify the binding of NLS-containing protein or molecularcomplex to the immobilized cellular receptor moiety as compared tobinding reactions in the absence of test compound. The absence of boundNLS-containing protein or molecular complex, or a reduction in thebinding of the NLS-containing protein or molecular complex to the solidsupport, in the presence of a test compound, indicates that the testcompound can be useful in inhibiting the importation of the specificNLS-containing protein or molecular complex into the nucleus.

The detection procedure may employ an antibody or a ligand thatrecognizes and binds the NLS-containing protein or a component of themolecular complex. Alternatively, if the molecular complex comprisesnucleic acids, such as the case of HIV-1 pre-integration complex,polymerase chain reaction may be employed to detect the presence ofspecific nucleic acid sequences on the solid support. Any appropriateisotopic and nonisotopic labels can be used in conjunction with thedetection procedure. Detection or measurement of the antibody, ligand oramplified nucleic acids is accomplished by standard techniques wellknown in the art. Those skilled in the art will be able to determineoperative and optimal conditions for the above-described techniques byemploying routine experimentation.

For example, a screening assay for alkyl aryl carbonyl compounds thatinactivate the NLS of HIV matrix antigen can be set up as follows. Aglutathione S-transferase-karyopherin α fusion protein (GST-karyopherinα) that binds NLS is used as the cellular receptor moiety. Thefusion-protein is immobilized onto glutathione Sepharose (Pharmacia) byincubation for 30-60 minutes at room temperature. Cytoplasmic extractsfrom cells that have been infected by HIV may be prepared bymechanically lysing infected cells in the presence of proteaseinhibitors, and removing the nuclei by centrifugation.

Alternatively, HIV MA and RT, and possibly other accessory proteins,such as Vpr, that are present in the pre-integration complex can beproduced by recombinant DNA methods, and reconstituted in vitro into acomplex for use in the assay. The cytoplasmic extract or thereconstituted MA/RT complex is incubated at room temperature with theGST-karyopherin α Sepharose beads in the presence of variousconcentrations of compounds of the invention, for an interval sufficientfor binding interactions to occur, for example, 30 minutes. At the endof the incubation, the beads are washed to remove any unbound material.To quantitate the binding of pre-integration complex to the beads, HIV-1DNA was isolated from the beads by SDS-proteinase K treatment withsubsequent phenol-chloroform extraction, and analyzed by polymerasechain reaction using primers specific for the pol gene, as described inBukrinsky, M. I., et al., 1995, J. Exp. Med. 181:735-745. Alternatively,the amount of MA or RT bound to the beads can be determined by animmunoassay using anti-MA or anti-RT sera. Compounds of the inventionthat can be used to block nuclear importation of HIV are indicated by areduction or inhibition of binding to the beads by the HIV-1preintegration complex or reconstituted MA/RT complex as compared to thecontrol.

6 EXAMPLES 6.1 Synthesis of Specific Compounds

Compound No. 2, FIG. 1A: A suspension of Compound No. 11(2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine) (0.284 g), wassuspended in 1:1 acetonitrile-tetrahydrofuran was treated with methyliodide (2 mL) and heated at 40-45° C. under a reflux condenser for 18hr. Cooling and filtration gave 0.35 g of2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium iodide, mp292° C.

2-Amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidine.A suspension of 21 g (49.3 mmole) of2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium iodide(compound No. 2, synthesized as described in section 6.1) in 1:1methanol/water (750 mL) at 60° C. was treated with excess 2N NaOH withcooling to maintain about 60° C. An additional 200 mL of water was addedand the mixture was cooled in ice and filtered to give 14.69 g2-amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidineas yellow crystals, mp 219-220°.

2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride(CNI-0294). CNI-0294 is the chloride salt of compound No. 2. The base2-amino-4-(3,5-diacetylphenyl)imino-1,4-dihydro-1,6-dimethylpyrimidine(14.35 g, 48 mmole) was dissolved in 500 mL of methanol and treated withHCl gas until precipitation appeared complete. Filtration gave 12.8 g ofwhite crystals with a faint yellowish tinge, mp 306.5-307.50°.

Compound No. 11 (CNI-1194): A suspension of 3,5-diacetylaniline (0.885g) in water (18 mL) was treated with 2-amino-4-chloro-6-methylpyrimidine(0.718 g) and concentrated HCl (0.42 mL) and heated at 90-100° C. for 30min. After cooling the mixture was treated with 10 mL of aqueous 1N KOH.The mixture was stirred for 10 min and the solid was filtered out,washed with water, and dried, to give 1.332 g of tan crystals.Recrystallization from ethyl acetate-2-methoxyethanol gave 1.175 g of2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine as light buffcrystals, mp 240-241° C.

Compound No. 12. A suspension of 3,5-diacetylaniline (0.531 g) in water(8 mL) was treated with cyanoguanidine (0.285 g) and conc. HCl (0.25 mL)and heated at reflux. After 6 hr the mixture was cooled and concentratedand 0.248 g of off-white solid was filtered out and dried to giveN-(3,5-diacetylphenyl)biguanide hydrochloride, mp 260-70° C. (dec).

Compound No. 13: A suspension of 3,5-diacetylaniline (1.95 g) in water(10 mL) was treated with 2-chloro-4,6-diamino-1,3,5-triazine (1.455 g)and concentrated HCl (0.1 mL) and heated at reflux for 20 min. Aftercooling the hydrochloride of Compound No. 13 separated as a whitepowder. This was filtered out, dissolved in 60 mL of boiling aqueous 75%methanol and treated with triethylamine (1.5 mL). On cooling, off-whiteflakes separated. Filtration and drying gave 1.79 g of2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine, mp 271-2° C.

Compound No. 14 (CNI-H1494):4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, Compound No. 15,(0.968 g) was suspended in acetone (5 mL) containing methyl iodide (2mL) was heated at reflux for 48 hr. Filtration after cooling gave 0.657g of 4-(3-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide asa white powder, mp 238-40° C.

Compound No. 15 (CNI-1594): A suspension of m-aminoacetophenone (2.7 g)and 2-amino-4-chloro-6-methyl-pyrimidine (2.87 g) in 40 mL water wastreated with 1.7 mL concentrated HCl and heated at reflux for 1 hour.Addition of 40 mL 1N KOH gave a light buff solid, which was filtered outand dried to give 3.8 g4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine, mp 196-98° C.

Compound No. 16: A suspension of 3,5-diacetylaniline (0.531 g) in water(10 mL) was treated with 6-chloropurine (0.464 g) and concentrated HCl(0.25 mL) and heated at reflux for 30 min. After cooling the mixture wastreated with 6 mL of aqueous 1N KOH. The mixture was stirred for 10 minand the solid was filtered out, washed with water, and dried, to give0.80 g of 6-[(3,5-diacetylphenyl)amino]purine, mp dec 340-350° C.

Compound No. 17 (CNI-1794): A suspension of p-aminoacetophenone (1.35 g)and 2-amino-4-chloro-6-methylpyrimidine (1.435 g) in 20 mL water wastreated with 0.85 mL conc HCl and heated at reflux for 1 hr. Addition of20 mL 1N KOH gave a light buff solid, which was filtered out and driedto give 2.28 g 4-(4-acetylphenyl)amino-2-amino-6-methylpyrimidine, mp194-196° C. Of this, 1.21 g was treated with methyl iodide (3 mL) indimethylformamide (15 mL) at room temperature for 42 hr. Dilution withethyl acetate and filtration gave 1.11 g4-(4-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide as awhite powder, mp 302-3° C.

Compound No. 45. (CNI-4594) A mixture of aniline (0.93 g) and2-amino-4-chloro-6-methylpyrimidine (1.44 g) in 36 mL water was treatedwith 0.84 mL conc HCl and heated at reflux for 1 hr. Addition of 20 mL1N KOH gave a light buff solid, which was filtered out, dried, andrecrystallized from ethyl acetate/2-methoxyethanol and ethylacetate/hexane to give 0.69 g 4-phenylamino-2-amino-6-methylpyrimidine,mp 179-180° C.

Compound No. 46. A suspension of4-phenylamino-2-amino-6-methylpyrimidine, Compound No. 45, (0.25 g) inethanol (4 mL) was treated with methyl methanesulfonate (0.090 g) andheated at reflux for 5 days. Additional methyl methanesulfonate (0.090g) was added and the mixture refluxed another 2 days. Concentration andrecrystallization from a mixture of methanol, ethyl acetate, andtert-butyl ethyl ether gave 0.10 g of4-phenylamino-2-amino-1,6-dimethylpyrimidinium methanesulfonate.

3,5-diacetylaniline (CNI-1894) was synthesized as per Ulrich et al.(1983, J Med Chem 27:35-40). Diacetylanilines substituted in otherpositions can be synthesized according to Ulrich et al. supra orMcKinnon et al. (1971, Can J Chem 49:2019-2022). All other startingmaterials were obtained from the Aldrich Chemical Co. Nuclear magneticresonance spectra and elemental analysis for all the compounds agreedwith expected values.

6.2 The Use of Compound No. 2 to Inhibit HIV Replication in PrimaryMacrophage Lines 6.2.1 Materials and Methods

Primary human monocytes were obtained from peripheral blood byFicoll-Hypaque centrifugation and adherence to plastic as describedpreviously. Gartner S. P., et al., 1986, Science 233:215. Briefly, afterFicoll-Hypaque (Pharmacia) separation, PBMCs were washed 4 times withDMEM (the last wash was done at 800 rpm to remove platelets) andresuspended in monocyte culture medium [DMEM supplemented with 1 mMglutamine, 10% heat-inactivated human serum, 1% penicillin+streptomycinmixture (Sigma)] at a density of 6×10⁶ cells/ml. Cells were seeded in24-well plates (1 ml per well) and incubated for 2 h at 37° C., 5% CO₂.Following incubation, cells were washed 3 times with DMEM to removenon-adherent cells and incubation was continued in monocyte culturemedium supplemented with 250 U/ml human M-CSF (Sigma). Cells wereallowed to mature for 7 days prior to infection with the monocyte-tropicstrain, HIV-1_(ADA). Nuovo, G. J., et al., 1992, Diagn. Mol. Pathol.1:98. Two hours after infection, cells were washed with medium andcultured in RPMI supplemented with 10% human serum. In experiments wherePCR analysis was performed, virus was pretreated with RNAse-free DNAse(Boehinger-Mannheim) for 2 h at room temperature and then filteredthough a 0.2 μm pore nitrocellulose filter prior to infection.

PBMCs were purified by Ficoll-Hypaque centrifugation and activated by 10μg/ml PHA-P (Sigma) and 20 U/ml recombinant human IL-2 (rhIL-2) in RPMI1640 supplemented with 10% FBS (HyClone). After 24 h incubation, cellswere washed and inoculated with HIV-1_(ADA) in RPMI 1640 supplementedwith 10% FBS. After a 2 h adsorption, free virus was washed away andcells were cultured in RPMI 1640 supplemented with 10% FPS and 20 U/mlrhIL-2.

Virus stock and infection. Macrophage-tropic strain HIV-1_(ADA) wasamplified in primary human monocytes and concentrated to produce stockwith TCID₅₀ of about 10⁵/ml. The concentration of HIV-1 was determinedby immunoassay of viral p24, concentration; using a conversion factor of1 ng / 200 HIV-1 particles.

6.2.2 p24 and RT Assay

For p24 assay, sequential 1:9 dilutions of culture supernatant wereprepared and analyzed by ELISA as suggested by the manufacturer(Cellular Products, Buffalo, N.Y.). For the reverse transcriptase (RT)assay, 10 μl of culture supernatant was added to 40 μl of reactionmixture (final composition was 50 mM Tris-HCl, pH 7.8; 20 mM KCl; 5 mMMgCl₂; 1 mM DTT; 0.1% Triton X-100; 0.2 OD/ml polyA; 0.2 OD/mloligo(dT)₁₂₋₁₈; and 40 μCi/ml ³H-dTTP (76 Ci/mmol, DuPont) and incubated2 hr at 37° C. 5 μl of the reaction mixture was then spotted onto the DE81 (Whatman) paper. Paper was air dried and washed 5 times with 5%Na₂HPO₄, followed by rinsing with distilled water. After air drying,paper was put on a Flexi Filter plate (Packard), covered withscintillation fluid and counted in a Top Count Microplate Counter(Packard). Results are expressed as counts per minute in 1 ml ofsupernatant (cpm/ml).

6.2.3 Results Dividing and Quiescent Cells

The cytotoxicity of Compound No. 2 was tested in monocyte cultures bytrypan blue exclusion assay or lactate dehydrogenase (LDH) release. Byboth assays, no cytotoxic effect was observed with concentrations of thecompound up to 10 μM (data not shown). Results presented in FIG. 2 showthe effect of various concentrations of Compound 2 on HIV-1 replicationin monocytes. From this experiment, we estimate the IC₅₀ for thiscompound between 0.1 and 1 nM. Similar and higher concentrations of thecompound were also tested on activated PBLs. The anti-viral effect ofthis compound was much less expressed in these actively dividing cellpopulations (FIG. 3). No anti-viral effect was detected when cultures ofreplicating cells were infected at the multiplicity of infection used toinfect monocytes.

6.2.4 AZT and Compound No. 2 in Combination

AZT is a drug that is routinely used to treat HIV-1 infected persons.However, two factors are known to diminish the effectiveness of AZT: itstoxicity and the emergence of resistant mutant strains of HIV-1. Theeffects of both of these factors can be reduced by administering asecond, synergistic HIV-1-inhibitory drug with AZT.

In view of these premises, the effects on HIV-1 replication in humanmonocyte cultures of the various concentrations of AZT, alone or incombination with 100 nM Compound No. 2, were tested using the protocolsof Sections 6.2.1 and 6.2.2. Drugs were added to the monocyte culturestogether with HIV-1 at about 10⁵ TCID / ml. The concentration of drugswas maintained on refeeding. HIV-1 replication was assessed by assay ofthe supernatant for reverse transcriptase activity. The results areexpressed as mean ±std. dev. (cpmx10⁻³) in Table V.

TABLE V Effects of Combined AZT/Compound No. 2 on HIV-1 infectedMonocyte Cultures day-7 day-11 [AZT] (−) No. 2 (+) No. 2 (−) No. 2 (+)No. 2 0 1.46 ± 0.43 0.37 ± 0.07 1.81 ± 0.75 0.72 ± 0.30 10 pM 0.92 ±0.21 0.15 ± 0.05 1.63 ± 0.81 0.18 ± 0.06 100 pM 0.79 ± 0.14 0.13 ± 0.041.34 ± 0.59 0.15 ± 0.06 1 nM 0.60 ± 0.28 0.04 ± 0.02 1.07 ± 0.49 0.09 ±0.03 10 nM 0.05 ± 0.02 0.03 ± 0.02 0.08 ± 0.03 0.07 ± 0.03

These results demonstrate that there is synergy between the AZT andCompound No. 2. The synergistic effects are most pronounced at the lowerdoses of AZT on day 11. For example, 10 pM AZT alone produces an about20% reduction in RT activity on day-11, 100 nM Compound No. 2 aloneproduces about a 60% reduction. Without synergy, the combination shouldproduce a 70% reduction (100×(1−(0.8×0.4)). Instead the observedreduction was 90%.

6.3 The Compounds of the Invention Do Not Block the Nuclear Importationof Essential Proteins in Cells 6.3.1 Direct Demonstration of theInhibition of HIV-1 Nuclear Importation by Compound No. 2

The effects of Compound No. 2 on the nuclear importation of HIV-1preintegration complexes can be directly measured by detecting thepresence of circularized duplex HIV-1 genomic DNA. These duplex circlescan be readily detected by PCR amplification using primers which spanthe junction of the circularized HIV-1 genome. Bukrinsky, M. I., et al.,1992, Proc.Natl.Acad.Sci. 89:6580-84.

Briefly, the efficiency of nuclear translocation was estimated by theratio between the 2-LTR- and pol -specific PCR products, which reflectsthe portion of 2-LTR circle DNA molecules as a fraction of the entirepool of intracellular HIV-1 DNA. Viral 2-LTR circle DNA is formedexclusively within the nucleus of infected cells and thus is aconvenient marker of successful nuclear translocation. Bukrinsky, M. I.,1992, Procd.Natl.Acad.Sci. 89:6580-84; Bukrinsky, M. I., 1993, Nature365:666-669.

PCR analysis of HIV-1 DNA: Total DNA was extracted from HIV-1-infectedcells using the IsoQuick extraction kit (Microprobe Corp., Garden Grove,Calif.). DNA was then analyzed by PCR using primer pairs that amplifythe following sequences: a fragment of HIV-1 (LTR/gag) that is the lastone to be synthesized during reverse transcription and thereforerepresents the pool of full-length viral DNA molecules; a fragment ofpolymerase gene (pol); a 2-LTR junction region found only in HIV-1 2-LTRcircle DNA molecules; or a fragment of the cellular a-tubulin gene.Dilutions of 8E5 cells (containing 1 integrated copy of HIV-1 DNA pergenome) into CEM cells were used as standards. Amplification productswere transferred to nylon membrane filters and hybridized to ³²p-labeledoligonucleotides corresponding to internal sequences specific for eachPCR amplification fragment, followed by exposure to Kodak XAR-5 film ora phosphor screen.

Quantitation of PCR Reactions: Bands of correct size revealed afterhybridization were quantitated with a PhosphorImager (MolecularDynamics) by measuring the total density (integrated volume) ofrectangles enclosing the corresponding product band. Efficiency ofnuclear translocation of HIV-1 DNA was estimated by measurement of theamount of 2-LTR circle DNA (N_(2-LTR)) relative to total viral DNA(N_(tot)) in each culture, indexed to the same ratio of appropriatecontrol cultures. Thus, Translocation Index=( N_(2-LTR)/N_(tot))/(C_(2-LTR)/C_(tot))×100.

Results: Primary human monocytes were infected with HIV-1_(ADA) in thepresence of 100 nM concentration of Compound No. 2 or without drugs(control). Half the medium was changed every 3 days, and drugs werepresent throughout the whole experiment. Cell samples were taken at 48and 96 hours post infection and the Translocation Index, relative to thedrug free control was determined. At both time points the TranslocationIndex was less than 10, indicating there was greater than 90% inhibitionof nuclear importation.

7 Pharmacokinetic and Toxicological Studies

This section describes in detail the techniques that were used to studythe toxicological and pharmacological properties of the compounds of theinvention.

7.1 Drug Analysis

Standard addition curves for each test compound were constructed byadding increased amounts of drug to mouse or human A⁺ plasma (LongIsland Blood Services; Melville, N.Y.). An equal volume of 10 mMtetramethylammonium chloride/10 mM heptane sulfonate/4.2 mM H₃PO₄(Buffer A) was added to the plasma sample, which was then loaded onto awashed 1 g cyanopropylsilane (or octadecylsilane for CNI-1894)solid-phase extraction column (Fisher Scientific). The column was washedwith 1.0 ml of water and then eluted with 1.0 ml of 10 mMtetramethylammonium chloride/10 mM heptane sulfonate/4.2 mM H₃PO₄/95%CH₃CN/5% H₂O (Buffer C). The eluted sample was reduced to dryness in arotary evaporator and resuspended in 1.0 ml Buffer A.

Two hundred μl of the resuspended sample was injected onto aHewlett-Packard model 1090 high performance liquid chromatography system(HPLC)(Wilmington, Del.) equipped with a photodiode arrayultraviolet/visible spectrophotometric detector, autosampler, andChemstation operating software. The column used was a 250×4.6 mm ZorbaxRX-C8 column (Mac-Mod Analyticals; Chadd's Ford, Pa.) kept at roomtemperature and run at 1.5 ml/min. The mobile phase used was Buffer Aand 10 mM tetramethylammonium chloride/10 mM heptane sulfonate/4.2 mMH₃PO₄/75% CH₃CN/25% H₂O (Buffer B), with all runs initiated at 10%Buffer B. A linear 30 min gradient to 60% Buffer B was then performed,followed by a 4 min reverse gradient to initial conditions. CompoundsCNI-0294, -1194, -1594, and -1794 were detected by ultravioletabsorbance at 300 nm, CNI-1894 at 240 nm, and pentamidine at 265 nm. Inthis assay system, the CNI test compounds have a linear response and aredetectable down to at least 19.5 ng per injection.

7.2 Toxicity Studies 7.2.1 Method

The doses of compounds of the invention found to be lethal to 50% of themice (LD₅₀) were determined by intraperitoneal injection of groups offive animals with increasing doses of each compound. CNI-0294 wasadministered from 0, 2, 10, 20, 40, 80, 160, 320, 640, 1280 mg/kg in 0.5ml of water/HCl; CNI-1594 at 0, 2.4, 5, 10, 20, 40, 80 mg/kg in 0.5 mlof water/HCl; CNI-1794 at 0, 20, 50, 80 mg/kg in 0.5 ml of water/HCl;and CNI-1894 at 0, 10, 20, 40, 80, 240, 480, 960 mg/kg in water/HCl. Allanimals were observed for visible signs of acute or long-term toxicity.The percentage of animals in each group which died were utilized tocalculate the LD₅₀ by non-linear curve fitting with the Enzfit software(Elsevier Bioscience; Cambridge, UK) programmed with the Chou equation(Chou 1976, J Theor Biol 39:253-276)).

7.2.2 Results

The compounds (FIG. 4A-E), were screened for toxicity via a modifiedLD₅₀ assay procedure as described above in an outbred strain of mice.The results are shown in Table VII as follows:

TABLE VI The toxicity of the CNI compounds, as measured by the medianlethal dose determined as described above. LD₅₀ ± standard deviationCompound (mg/kg) 0294  587.77 ± 65.79 1194 >160* 1594  49.04 ± 0.08 1794 48.93 ± 0.12 1894 258.64 ± 1.37 *Higher doses were not tested due tolimiting amounts of the compound.

CNI-0294 was found to be very well tolerated (see Table VI), with noovert signs of toxicity detectable at doses approaching the LD₅₀. Theother compounds in the CNI series were designed to allow forstructure-function relationships with respect to activity and toxicity.CNI-1194, which differs from CNI-0294 only by the lack of a methyl groupon the heterocyclic nitrogen, was also well tolerated, with a high LD₅₀(Table VI). However, CNI-1594, which is similar to CNI-1194 plus theomission of one of the acetyl groups on the benzene rings, wasappreciably more lethal (Table VI). This toxicity was immediate, withdeath occurring in minutes and the animals displaying signs of acuteneurotoxicity. CNI-1794, which is identical to CNI-1594 except that thesingle acetyl group is moved para to the heterocyclic substituent, hadan LD₅₀ identical to that for CNI-1594 (Table VI). CNI-1894, which issimilar to CNI-0294 and -1194 but lacks the heterocyclic ring, was alsoreasonably well tolerated. Animals dosed with large amounts of CNI-1894died 2-3 days post injection, and showed no sign of any immediatetoxicity. Based on the above observation, it is concluded that thepresence of the heterocyclic ring in the compounds of the inventionplays only a small role in determining toxicity, while the presence oftwo acetyl groups on the benzene ring is very important. Therefore, apreferred compound of the invention showing low toxicity contains twoacetyl groups on the benzene ring.

7.3 Pharmacokinetic Studies 7.3.1 Methods

Female ND4 Swiss Webster mice (21-24 g) were obtained from HarlanSprague Dawley (Indianapolis, Ind.) and randomly placed in groups offive in cages with free access to food and water. Each group of animalsreceived 50 mg/kg of CNI-0294, -1194, or -1894, or 20 mg/kg of CNI-1594in a volume of 0.5 ml. Compound CNI-0294 was administeredintraperitoneally or by oral gavage as a solution in water or asuspension in 10% DMSO/peanut oil. The other CNI compounds wereadministered intraperitoneally or by oral gavage as a solution in watertitrated with sufficient HCl to dissolve the drug. At various timepoints, ranging from 5 min to 4 days, a single group of animals waseuthanized by carbon dioxide inhalation and bled by cardiac punctureusing heparin as an anticoagulant. The blood from the five mice in thegroup was pooled and centrifuged at 14000×g for 10 min. The volume ofplasma was measured, and equal volume of Buffer A added, and the mixtureextracted and analyzed as described above, except that the dried eluateswere resuspended in 200 μl Buffer A and 100 μl was injected onto thehigh performance liquid chromatography (HPLC) system.

As inspection of the blood concentration-time curves for a single i.p.injection showed a typical biphasic appearance, standard methods ofpharmacokinetic measurement were employed (1982, Gibaldi et al.,Pharmacokinetics. Marcel Dekker, New York). The area under the plasmaconcentration-time curve (AUC) was determined, and bioavailability wasmeasured as AUC_(oral)/AUC_(i.p.) A and B represent the zero timeintercept of the distribution and elimination phases respectively, and αand β the respective slopes of the phases multiplied by 2.303. Thet_(1/2α) and t_(1/2β) are calculated half-lives of the drug in eachphase (0.693/α and 0.693/β respectively). The volume of distribution(V_(D)) was calculated as dose/B, and the total clearance rate(Cl_(tot)) calculated as β*V_(D). C_(max) and t_(max) are the maximalplasma concentration and time of this measurement, respectively.

7.3.2 Results

As judged by the plasma concentration-time curves from a singleintraperitoneal injection, each compound in the CNI series had similarpharmacokinetic properties despite the structural differences. Thekinetic parameters are summarized in Table III and a typical pattern isshown for CNI-1194 in FIG. 5. The drugs were rapidly absorbed, with themaximal plasma concentration reached in 5-15 min, and also had a rapiddistribution phase, with a t_(1/2α) of 0.32-0.62 hr. Differences werefound to occur in the maximal plasma concentration and parametersrelated to the elimination phase. CNI-0294 achieved the highest maximalplasma level for a single 50 mg/kg i.p. injection, with 18.76 μg/ml, andCNI-1894 was very similar with a value of 13.43 μg/ml. As CNI-1194 hadan appreciably lower maximal plasma level and a slower t_(max) whencompared with CNI-0294, it appears that the presence of the methylsubstituent on the heterocyclic nitrogen enhances drug absorption fromthe peritoneum. A comparison of CNI-1194 and CNI-1594 implied that thenumber of acetyl groups had little effect on drug absorption. The valuesrelating to elimination (β, B, t_(1/2β), V_(d), Cl_(tot)) were found tovary, but no clear structural relationship could be discerned. All thecompounds, except CNI-1894, were undetectable in plasma after 24 hr andapproached the limit of detection after 5-6 hr. Therefore, as a generalproperty, the compounds of the invention are absorbed and eliminatedrapidly. A preferred compound of the invention has a methyl substituenton the heterocyclic ring nitrogen at position 1 and possesses enhancedabsorption from the peritoneum.

Experiments were also performed with CNI-0294 and -1194 to evaluaterelative bioavailability. By comparing the AUC_(oral) against theAUC_(i.p.) for a single 50 mg/kg dose, CNI-0294 was found to have 6%relative bioavailability and CNI-1194 15%. The maximal plasma level was0.4 μg/ml for CNI-0294 and 0.35 μg/ml for CNI-1194, and the drugs weredetectable in plasma for at least 6 hr (see FIG. 5).

7.4 Metabolic Studies

During the analysis of the plasma samples for the pharmacokineticparameters, a number of additional HPLC peaks were detected whichincreased and decreased over time. Extra peaks of this nature were seenin samples from each of the CNI series as shown in FIGS. 8A-8D. As itwas possible that these peaks represented metabolites of the CNIcompounds, the compounds of the invention were screened in a simplemodel of primary metabolism.

7.4.1 Method

Several female ND4 Swiss Webster mice were euthanized by carbon dioxideinhalation and the livers excised and rinsed with ice cold phosphatebuffered saline (pH 7.4). The livers were minced, gently homogenized in50 mM phosphate buffer (pH 7.4) with a Dounce homogenizer, andcentrifuged at 9600×g for 20 min. The post-mitochondrial supernatant waskept, glycerol added to 20%, and frozen at −70° C. in 1.0 ml aliquotsuntil used. For each incubation, 1.0 ml of a 1.0 mg/ml drug solution wasadded to 3.0 ml of 50 mM phosphate buffer (pH 7.4), 1.0 ml of 2 mg/mlNADPH in 50 mM phosphate (pH 7.4), and 1.0 ml of the post-mitochondrialsupernatant. Five hundred μl of each incubate was then immediatelytransferred to an ice-cold tube to provide the zero-time sample, andaddition 500 μl aliquots removed to ice-cold tubes at 8, 15, 30, and 60min. The samples were then extracted, and analyzed by HPLC as describedin section 7.1. Control incubations were also performed where drug orpost-mitochondrial supernatant was omitted. An incubation usingpentamidine was performed to confirm microsomal activity (Berger et al.,1992, Antimicrob. Ag. Chemother. 36:1825-1831). Peaks in the CNIcompound incubations which increased over time, and were not present incontrol samples lacking the enzyme preparation were treated as putativemetabolites.

7.4.2 Results

Using post-mitochondrial supernatants of homogenized mouse livers as asource of enzyme, the drugs were incubated in the presence of NADPH. Asdescribed in Berger et al. supra, pentamidine was used as a positivecontrol, and the seven, expected, primary metabolites were detectable,confirming the activity of the enzyme preparation. Extraction andanalysis of the CNI incubates showed the presence of numerous, putativemetabolite peaks that were not present in negative control incubations(FIG. 6). Incubation of CNI-0294, -1594, or -1194 was found to producethree minor and one major metabolite and CNI-1894 had one minor and onemajor metabolite. The major metabolite was found to elute 0.9-1.2 mincloser to the solvent front for CNI-0294, -1194, and -1594, suggestingthat the same position was being altered in each of these compounds. Themetabolic conversion in the post-mitochondrial supernatant system wasconsiderable, with 43.5% of CNI-0294, 65.19% of CNI-1194, 11.74% ofCNI-1594, and 17.28% of CNI-1894 altered during the course of a 60 minincubation (as judged by peak area). These results indicated thatappreciable metabolism of the compounds of the invention should occur invivo.

Re-examination of the plasma samples confirmed that the several of theunknown plasma peaks seen in FIGS. 6A and 6B corresponded to theputative metabolites in FIGS. 7A-7D. However, the metabolic model systemdid not produce all the unknown peaks seen in the plasma samples. Inparticular, a plasma peak eluting at 11-14 min was seen with all thecompounds in vivo, but not seen at all in the in vitro test system. Aswas evident from the plasma time-course samples, there appeared to be alarge amount of metabolic conversion in vivo of all of the compounds,regardless of the route of administration.

7.5 Conclusions

The toxicity, pharmacokinetics, and metabolism of the novel arylenebis(methylketone) compounds of the invention, and several novelanalogues thereof likewise of the invention were examined in mice. Witha median lethal dose of 587.77 mg/kg, CNI-0294 was well tolerated whenadministered intraperitoneally. Analogues which also had two acetylgroups on the phenyl moiety were also well tolerated, with median lethaldoses exceeding 160 mg/kg i.p. All visible toxic reactions appeared tobe rather delayed (generally 2-3 days post injection). While no biopsysamples were taken, such a delay would be consistent with organ damageby very high doses these compounds. Compounds which had only one acetylgroup were found to be more toxic, with median lethal doses of48.93-49.04 mg/kg i.p. While the visible symptoms following injection ofCNI-1594 or -1794 suggested a lethal neurotoxicity, the structuraldifferences between the two drugs indicate that antagonism of anendogenous neurotransmitter is unlikely.

In test animals, all of the compounds possessed very rapidpharmacokinetic properties, with the plasma maximal concentration, forintraperitoneal injection, being reached in 5-15 min, and 15-60 min fororal dosing. For CNI-0294, a plasma maximal concentration of 18.76-18.93μg/ml was reached after injection of 50 mg/kg i.p. The other compoundstested achieved lower maximal plasma levels (1.9-13.43 μg/ml). Thehalf-life of the distribution phase (t_(1/2α)) was 0.32-0.62 hours, andthat for the elimination phase (t_(1/2β)) was 3.65-23.10 hours. All ofthe kinetic parameters are consistent with drugs that are very rapidlycleared from the plasma and are not retained in tissues for a longperiod of time. Both CNI-0294 and -1194 were orally absorbed, with arelative bioavailability of 6 and 15 percent respectively. This latterfeature is very favorable for continued development of these compoundsas anti-infective agents, particularly as antiviral and antiparasiticagents, and more particularly as anti-retroviral and anti-protozoalagents, and yet particularly as anti-HIV agents and antimalarials. Thetoxicity, kinetic, and bioavailability data suggest that frequent, high,oral doses of the CNI-0294 can safely maintain therapeutically effectiveplasma concentration.

Metabolism of the drugs was assessed in a mouse liver post-mitochondrialsupernatant system, and extensive metabolism was discovered(11.74-65.19% metabolized during, a 60 minute incubation). Examinationof plasma samples showed that there was considerable in vivo metabolism,with at least 4-6 metabolites easily detected during the first 3 hoursfollowing i.p. administration of the test compounds. The levels ofmetabolite rapidly exceeded plasma concentrations of the parentcompound. The HPLC retention times indicated that the compounds werelikely altered in the same positions. In addition, the metabolites, likethe parent compounds, appeared to have very rapid plasma kinetics.

8 EXAMPLE

Demonstration of Anti-Malarial Activity

8. 1 The Compounds have Anti-Malarial Activity in Vitro 8.1.1 Method

The antimalarial activity of the compounds was determined essentially asdescribed in Desjardins et al. supra. Fifty μl of various concentrationsof a compound of the invention, chloroquine, or pyrimethamine were addedto the wells of microtiter plates, followed by 200 μl of ring-stage,synchronized, P. falciparum-infected erythrocytes (finalhematocrit=1.5%, final parasitemia=1-5%). The plates were incubated for24 hr in a candle jar kept at 37° C., and then 25 μl of[³H]-hypoxanthine (Amersham, Arlington Heights, Ill.; 2.5 μl Ci/well)was added. The plates were then incubated for a further 24 hr, beforeharvesting onto Unifilter-96 GF/C filter-microplates (Packard; Meriden,Conn.). Twenty-five μl of Microscint scintillation fluid (Packard) wasadded to each well of the filter-microplate, which was subsequentlycounted in a Top-count microplate scintillation counter (Packard). Thepercent of [³H]-hypoxanthine uptake relative to controlinfect-erythrocytes was used to determine the IC₅₀ value for thecompounds by non-linear regression for LD₅₀ determination.

8.1.2 Results

Using the hypoxanthine-incorporation method for assessing Plasmodiumfalciparum growth in vitro as described above, CNI-0294 was found tohave considerable anti-malarial activity (Table VII).

TABLE VII The antimalarial activity of CNI-0294, chloroquine, andpyrimethamine in vitro against several Plasmodium falciparum clones. Themedian inhibitory concentration was determined as described above.Pyrimethamine IC₅₀ CNI-0294 IC₅₀ Clone Chloroquine IC₅₀ (μM) (μM) D10 26.99 ± 2.42* 170.70 ± 24.60  4.00 ± 0.41 Dd2 122.54 ± 7.26 103.70 ±9.79  3.52 ± 0.10 FCR-3 104.68 ± 9.98 0.04 ± 0.01 3.09 ± 0.30 HB3  6.73± 0.16 8.97 ± 2.75 1.79 ± 0.27 W2mef 143.79 ± 13.30 17.81 ± 13.46 2.29 ±0.22 *Each value is ± standard deviation (n = 4 for chloroquine andCNI-0294, and n = 2 for pyrimethamine).

The median inhibitory concentration (IC₅₀) for CNI-0294 was calculatedto be 1.79-4.00 μM for a series of cloned parasites which have differentsensitivities to chloroquine or pyrimethamine (Table VII).

The Dd2 clone of P.falciparum, which was both chloroquine andpyrimethamine resistant, was utilized to compare the antimalarialactivity of the remaining CNI compounds (Table VIII).

TABLE VIII The antimalarial activities of the CNI compounds against thechloro- quine- and pyrimethamine- resistant P. falciparum clone Dd2. Themedian inhibitory concentration was determined as described above.Compound IC₅₀ ± standard deviation (μM) 0294  3.67 ± 0.57* 1194 20.27 ±1.62 1594 23.73 ± 0.59 1894 >200** 4594 25.11 ± 0.72 *n = 4 for all. TheCNI-0294 replicates were independent of those shown in Table VII.**Highest concentration tested.

In independent measurements, CNI-0294 agreed well with the results inTable VII, and CNI-1194 was found to be approximately 5-fold lessactive. This difference suggested that the heterocyclic methyl group isrequired for maximal activity. CNI-1594 had an IC₅₀ equal to that forCNI-1194 or CNI-4594 demonstrating that loss of one or both of theacetyl groups can have little effect on the antimalarial activity.CNI-1894, however, was inactive at the highest concentration tested.

8.2 The Compounds have Anti-Malarial Activity in Vivo 8.2.1 Method

The antimalarial activity of CNI-0294 in vivo was assessed by infectingfemale ND4 Swiss Webster mice with 100 μl of Plasmodium berghei NYU-2infected mouse erythrocytes (50% parasitemia) by intraperitonealinjection. The animals were subsequently injected intraperitoneally onceper day on days 1-4 of the infection with 0.5 ml water or 0.5 ml of 50mg/kg CNI-0294 in water. Four hours after the final injection, smallblood samples were taken from the tail, and thin smears stained withDif-Quick (Baxter, Miami, Fla.). The parasitemia of control and treatedanimals was enumerated by inspection of at least 1000 erythrocytes ineach animal.

8.2.2 Results

As the CNI-0294 IC₅₀ for P.falciparum was in the range achieved forapproximately one hr following a single i.p. injection of 50 mg/kg inmice, the compound was also screened in vivo in mice infected withPlasmodium berghei. Utilizing the four day suppression test, whereparasitemia is enumerated following four daily injections of the testcompound (in this case 50 mg/kg i.p.), CNI-0294 was found tosignificantly (P≦0.01) lower the parasitemia by 10-fold (FIG. 9).

8.3 Conclusions

As indicated in Table VII, CNI-0294 was effective against various clonesof P. falciparum. The consistency in CNI-0294 IC₅₀ over such a range ofchloroquine and pyrimethamine IC₅₀'s suggested that CNI-0294 had adifferent mechanism of action than either of these establishedantimalarials.

While daily 50 mg/kg injections i.p., for 4 days, were found to stronglysuppress P. berghei infection in mice, these animals were not completelycured during this course of treatment. The difference between these invivo results and the more striking P.falciparum in vitro results arelikely due to the kinetic and metabolic properties of the compound. Invitro, the parasites are exposed to a constant level of the drug for 48hr, with no source of host metabolizing enzymes. In the case in vivo,the single, daily i.p. injection only provides therapeutic plasmaconcentrations for approximately one hour and there is considerablemetabolism to compounds which may have reduced anti-plasmodial activity.In light of these observations, one of ordinary skill in the art wouldbe able to further optimize the dosing regimens.

9 EXAMPLE

Mechanism of Inhibition of HIV-1 Nuclear Translocation by Compound No. 2

The following experiments demonstrate the inhibitory mechanism ofcompound No. 2 (also known as CNI-0294 or CNI-H0294) on HIV nuclearlocalization, which is based on the inactivation of the nuclearlocalization sequence (NLS) of the HIV matrix antigen (MA) in thepresence of HIV reverse transcriptase (RT). The results described hereinprovide a basis for the development of a novel class of antiviralcompound that inhibit nuclear localization and that are selectiveagainst specific NLS-containing proteins or molecular complexescomprising NLS-containing protein.

9.1 Materials and Methods

Infection with Mutant HIV-1 or HIV-like Pseudovirions

H9 cells were infected with HIV-1 or HIV-like pseudovirions (Haffar, etal., 1990, J. Virol., 64:2653-2659) at a multiplicity adjusted accordingto p24 content (50 ng p24 per 106 cells). The MA NLS virus containssubstitutions of isoleucine residues for lysines in positions 26 and 27of MA in an NLHX backbone (Westervelt., et al., 1992, J. Virol.66:2577-2582), thus inactivating the NLS. The Vpr⁻ virus has theinitiating ATG of the vpr gene changed to GTG, thus abolishingexpression of this gene. The ΔMA NLS pseudovirions have leucinesubstituting for lysine in position 28 of MA. This mutation abrogatesnuclear translocation of HIV-1 gag RNA in growth-arrested H9 cellsinfected with pseudovirions. After a 1 hour absorption, excess virusesor pseudovirions were washed away, and cells were incubated for anadditional 2-3 hour period at 37° C. prior to analysis.

Preparation of Cytoplasmic Lysates

Cytoplasmic extracts were prepared by lysing cells in cold extractionbuffer (10 mM KCl, 10 mM Tris-HCl [pH 7.6], 0.5 mM MgCl₂, 1 μg/ml eachof leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl fluoride[PMSF]) by 20-30 strokes of a Dounce homogenizer under the control ofphase-contrast microscopy. After removal of nuclei, cytoplasmic extractwas cleared by centrifugation at 15,000 g for 10 min.

Analysis of Binding of Nucleoprotein Complexes to Karyopherin α

Cytoplasmic extracts prepared from HIV-1- or pseudovirion-infected H9cells were adjusted to 0.14 M NaCl, 0.1% Tween 20 and precleared withglutathione-Sepharose beads for 30 min. at room temperature. Karyopherinα was expressed as a fusion protein with glutathione S-transferase (GST)which can bind glutathione beads in solution. GST-karyopherin αimmobilized on Sepharose beads was then added (about 50 μg ofimmobilized karyopherin α per extract from 108 infected cells) and themixture was incubated at room temperature for another 30 min. Beads werethen pelleted by centrifugation and washed 3 times with PBS supplementedwith 0.1% Tween 20, 1 μg/ml each of leupeptin and aprotinin, and 1 mMPMSF. HIV-1 DNA was isolated from the beads by SDS-proteinase Ktreatment with subsequent phenol-chloroform extraction, whilepseudovirion gag RNA was isolated by RNazol (Biotecx Laboratories Inc.).

Analysis of CNI-H0294 Interaction with HIV-1 Proteins in Solution

0.28 nanomoles of recombinant MA or RT [p66/p51 dimer were mixed with 20nmol of [¹⁴C]CNI-H0294 (specific activity 5×10⁴ cpm/nmol) and incubated2 hr at room temperature in 40 μl of binding buffer (PBS supplementedwith 1% BSA, 0.1% Tween 20, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mMPMSF). Sheep anti-MA or rabbit anti-RT sera (both obtained from NIH AIDSResearch and Reference Reagent Program) or pre-immune control sera werethen added (at 1:100 dilution) and incubation continued for another 1 hrat room temperature. Immune complexes were precipitated with proteinG-agarose, washed, and then eluted with 0.1 M glycine, pH 2.8.Radioactivity of the eluate was measured in a scintillation counter.

Analysis of CNI-H0294 Interaction with HIV-1 Pre-integration Complexes

Cytoplasmic lysates prepared from HIV-1-infected cells were treated with10 μM of [¹⁴C]-labeled CNI-H0294 (specific activity 5×10⁴ cpm/nmol) in 1ml extraction buffer adjusted to 0.14 M NaCl. Sodium borohydride wasthen added to a final concentration 10 mM and samples were incubated 1hr at room temperature prior to immunoprecipitation to reduce doublebonds of Schiff bases to an irreversible secondary amine.Immunoprecipitation was performed as described above, but beads werestringently washed three times with PBS supplemented with 0.1% SDS, 1%sodium deoxycholate, 1 μg/ml each of aprotinin and leupeptin, and 1 mMPMSF.

9.2 Results

CNI-H0294 reacts with adjacent lysines in the NLS, thus making itcapable of neutralizing NLSs on many different proteins. Interestingly,CNI-H0294 exhibited remarkably low cytotoxicity in monocyte and Tlymphocyte cultures in vitro (50% toxic dose >1 mM) and in vivo in mice(LD50=590 mg/kg, see Table VI). These results suggest that the molecularmechanism of MA NLS inactivation by CNI-H0294 is very specific. Indeed,this compound, did not block nuclear import of nucleoplasmin-coated goldparticles, nor of BSA with conjugated NLS peptides that mimic the NLS ofSV40 large T antigen.

CNI-H0294 Inhibits Interaction Between HIV-1 Pre-Integration Complexesand Karyopherin α, but does not Affect Binding of Karyopherin α toPseudovirion-derived Nucleoprotein Complexes

The initial step in the process of nuclear import is binding ofkaryopherin α (also termed NLS-receptor/importin) to an NLS. Resultspresented in FIG. 10A demonstrate that wild-type HIV-1 pre-integrationcomplexes bound to GST-karyopherin α immobilized onglutathione-Sepharose beads (lane 1). Mutant pre-integration complexesthat lack Vpr (MA NLS⁺Vpr⁻, lane 3) bound with reduced efficiency, whilebinding of the complexes with mutated MA NLS (MA NLS⁻Vpr⁺, lane 2) waseven more impaired. Pre-integration complexes that lack Vpr and aremutant in MA NLS (MA NLS⁻Vpr⁻) did not bind to karyopherin α (lane 4).These results are consistent with the analysis of MA and Vpr binding tokaryopherin α which demonstrated that while Vpr can bind weakly tokaryopherin α, its main role is to enhance the MA NLS-karyopherin αinteraction.

To facilitate analysis of HIV-1 nuclear translocation and of themechanism of drug effects on this process, a simplified model of theHIV-1 pre-integration complex was used which comprises a minimal numberof non-essential proteins. This model employs gag-env pseudovirionswhich exhibit an HIV-like core but are composed exclusively of Gag (MA,CA, NC, p6) and Env (gp41 and gp120) proteins. These pseudovirionspackage HIV-1 gag RNA and translocate this RNA into the nucleus of aninfected cell in a manner similar to the behavior of HIV-1pre-integration complexes. Results presented in FIG. 10B demonstratethat karyopherin α binds nucleoprotein complexes formed inpseudovirion-infected CD4+T cells (lane 3). Binding required afunctional MA NLS as mutation of the NLS (FIG. 10A, lane 1) orpre-treatment of nucleoprotein complexes with polyclonal anti-MAantibodies (lane 2) greatly diminished binding to karyopherin α. Thus,it is concluded that pseudovirion-derived nucleoprotein complexesinteract with karyopherin α in a manner similar to HIV-1 pre-integrationcomplexes.

The effect of CNI-H0294 on the interaction between karyopherin α andHIV-1 versus pseudovirion nucleoprotein complexes was examined. It wasfound that CNI-H0294 inhibited in a dose-dependent manner binding ofkaryopherin α to HIV-1 pre-integration complexes (FIG. 10C, top panel).Quantitation on a Phosphorimager demonstrated that 0.1 μM and 1 μM ofCNI-H0294 reduced karyopherin α/HIV-1 binding 8- and 25-fold,respectively. These results explain the inhibition of HIV-1 nuclearimport by the compound and correlate well with the dose response curveobtained when HIV-1-infected monocyte cultures were treated withCNI-H0294. Surprisingly, CNI-H0294 did not inhibit binding ofkaryopherin α to pseudovirion-derived nucleoprotein complexes (FIG. 10C,bottom panel) or to purified recombinant MA. These results suggest thatthe mechanism of CNI-0294 inhibition requires a factor(s) present in theHIV-1 pre-integration complex but absent from pseudovirion-derivedcomplexes.

Structure-activity Relationships within the CNI-H Group of Compounds

To further investigate the mechanism of action of CNI-H0294, thestructure-activity relationships within compounds of the invention wereexamined (Table IX).

Table IX Structure-function Analysis of Anti-HIV Activity of CNICompounds

CNI compounds were added at various concentrations (10 pM to 10 nM) tocultures of primary human monocytes together with HIV-1_(ADA) and werepresent throughout the entire experiment. A 50% inhibitory concentration(IC₅₀) was determined at day 9 after infection. Some compounds did notachieve 50% inhibition at maximal concentration yet exhibited anti-HIVactivity; in these cases the results are present as >10 μM.

Compound Structure IC₅₀ CNI-H0294

50 nM CNI-H1894

>10 μM CNI-H1494

1 μM CNI-H3094

>10 μM

Absence of the reactive carbonyl groups (compounds CNI-H1494 andCNI-H3094) or the pyrimidine side chain (compound CNI-H1894) resulted ina dramatic decrease of the drug's potency. As the carbonyl groups weredesigned to react with lysine residues within MA NLS, it was notsurprising that their absence decreased the drug's activity. Incontrast, a role for the pyrimidine side chain was unexpected, andsuggested that this side group may be involved in binding CNI-H0294 tothe pre-integration complex.

CNI-H0294 Binds to RT

Binding of CNI-H0294 to RT or MA was tested in vitro using [¹⁴C]-labeledCNI-H0294 and recombinant RT and MA proteins (FIG. 11). Specificimmunoprecipitation was used to quantify the amount of bound CNI-H0294.Preliminary experiments showed that both anti-RT and anti-MA reagentsspecifically recognized and immunoprecipitated RT and MA, respectively.As shown in FIG. 11, about 17,000 cpm, or 0.34 nmol of CNI-H0294(specific activity 50,000 cpm/nmol) were immunoprecipitable fromincubations of drug with 0.28 nmol RT, suggesting that CNI-H0294 bindsto RT in a 1:1 molar ratio. The specificity of this interaction wasfurther confirmed by immunoprecipitation experiments using coldCNI-H0294 to compete out precipitable counts associated with labeleddrug (FIG. 11). No binding was observed with recombinant MA, and noradioactivity was precipitated by immune sera if the recombinant proteinwas omitted from the reaction mixture. In similar experiments, thebinding of CNI-H0294 to Vpr nor to integrase, two other proteins knownto be present within the HIV-1 pre-integration complex, were detected.These experiments established that CNI-H0294 bound directly to RT, butnot to other proteins of the HIV-1 pre-integration complex. Of interest,CNI-H0294 did not significantly inhibit reverse transcription of HIV-1in infected cells, nor did it block in concentrations up to 50 μM theenzymatic activity of HIV-1 RT in vitro, suggesting that an effect on RTactivity cannot account for the anti-viral action of the compound.

Binding to RT is Critical for the Anti-HIV Activity of CNI-H0294

The role of CNI-H0294/RT interaction in the drug's activity was analyzedin experiments with compound CNI-H3094. As CNI-H3094 does not havereactive carbonyl groups but contains the active pyrimidine side chain(see Table IX), it could effectively compete with CNI-H0294 for bindingto the same site on the HIV-1 pre-integration complex, albeit it did notinhibit nuclear import of HIV-1. FIG. 12A shows that unlabeled CNI-H3094inhibits binding of [¹⁴C]-labeled CNI-H0294 to RT in a dose-dependentmanner. Likewise, CNI-H3094 restored binding of HIV-1 pre-integrationcomplexes to karyopherin α in the presence CNI-H0294 (FIG. 12B). A5-fold excess of CNI-H3094 (FIG. 12B, lane 4) reduced significantly theinhibitory effect of CNI-H0294 on binding of HIV-1 pre-integrationcomplexes to karyopherin α, and a 10-fold excess (lane 5) completelyeliminated the inhibitory effect. In a control experiment, CNI-H3094 didnot inhibit binding of HIV-1 pre-integration complexes to karyopherin α(FIG. 12B, lane 1); this correlates with the compound's lack of anti-HIVactivity (Table IX). Finally, CNI-H3094 eliminated the inhibitory effectof CNI-H0294 on HIV-1 replication in monocyte cultures (FIG. 12C). Theseresults confirm the critical role of CNI-H0294/RT interaction in thedrug's mechanism of action and also show a direct correlation betweenthe drug's binding to RT, inhibition of HIV-1/karyopherin α interaction,and repression of viral replication.

CNI-H0294 Inactivates the NLS of MA without Disrupting MA Associationwith the HIV-1 Genome

The results presented herein indicate a direct role for RT in theanti-HIV effect of CNI-H0294 and provide a molecular explanation for thehigh specificity of the compound. However, these results do not explainhow CNI-H0294 prevents binding of HIV-1 pre-integration complexes tokaryopherin α, as RT does not bind to directly to karyopherin α. Onepossibility was that binding of CNI-H0294 to RT disrupts thepre-integration complex and causes dissociation of MA from HIV-1 cDNA.

To test this hypothesis, cytoplasmic lysates of HIV-1-infected H9 cellswere treated with CNI-H0294 and mixed with immobilized karyopherin α orsubjected to immunoprecipitation with antibodies that bind MA. AlthoughCNI-H0294 blocked the interaction of the pre-integration complexes withkaryopherin α (FIG. 13A, lane 2), it did not prevent immunoprecipitationof viral DNA with anti-MA serum (FIG. 13A, lane 3); thus MA was stillassociated with HIV-1 cDNA but lost its ability to bind karyopherin α.As binding of pre-integration complexes to karyopherin α is controlledmainly by the MA NLS (FIG. 10A and 10B), these results indicate thatCNI-H0294 neutralizes the NLS activity of MA, either directly (throughchemical modification) or indirectly (e.g. by steric hindrance).

To discriminate between these two possibilities, cytoplasmic extracts ofHIV-1-infected cells were treated with [¹⁴C]-CNI-H0294 and then withsodium borohydride, to reduce the reversible Schiff bases hypothesizedto form between the compound and the lysines of MA NLS and convert theattached drug molecules to irreversible adducts. MA was thenimmunoprecipitated with specific serum in a buffer containing of 0.1%SDS and 1% sodium deoxycholate which disrupts weak protein-drug andprotein-protein interactions in the pre-integration complex withoutdisrupting covalent bonds. Under these conditions, a significant amountof radioactivity was immunoprecipitated by anti-MA serum (FIG. 13B), incontrast to results obtained with recombinant MA (FIG. 11). Theseresults corroborated requirement for RT for the drug's effect andsuggested that the CNI-H0294 had been covalently linked to MA byborohydride treatment. Without borohydride treatment, no radioactivitywas immunoprecipitated with MA. In control experiments, no radioactivitywas precipitated by anti-IN serum, or by anti-MA serum from thecytoplasmic extract of cells infected with pseudovirions (FIG. 13B)which lack RT and thus do not bind CNI-H0294 (FIG. 11).

CNI-H0294 Inhibits MA NLS⁻, but not Vpr-mediated Binding of HIV-1Pre-integration Complexes to Karyopherin α

Because of the role for MA NLS and Vpr in HIV-1 nuclear import, theeffects of CNI-H0294 on the interaction between karyopherin α andpre-integration complexes derived from viruses that carry mutations inVpr (Vpr⁻), MA NLS (MA NLS⁻), or both were investigated (FIG. 11). Theseviruses (except for MA NLS⁻Vpr⁻double mutant which was slightlyattenuated) entered target cells and reverse transcribed their genomewith similar efficiencies (FIG. 14A). The presence of CNI-H0294diminished binding of karyopherin α to wild-type (wt) (FIG. 14B, lanes1, 2) and Vpr⁻complexes (lanes 3, 4) by 95% but had no effect on bindingto MA NLS⁻ complexes (lanes 5 and 6) which is only 1-5% of that observedwith MA NLS+ complexes. The inability of CNI-H0294 to block binding ofMA NLS⁻ complexes to karyopherin α can be explained by the lack of aconsensus NLS in Vpr and that Vpr binds to karyopherin α in anNLS-independent manner.

Results presented hereinabove reveal the molecular mechanism of actionof CNI-H0294 that specifically target nuclear import of HIV-1. Themechanism by which CNI-H0294 can inactivate the MA NLS and thus preventnuclear import of HIV-1 is depicted in FIG. 15. According to theinvention, the compound first binds to HIV-1 pre-integration complexesvia RT and then forms reversible Schiff bases with contiguous lysines inan adjacent MA NLS. This interaction prevents binding of karyopherin αto the MA NLS and significantly inhibits nuclear translocation of theHIV-1 pre-integration complex. The results with pseudovirion-derivednucleoprotein complexes indicate that formation of the functionalcomplex capable of binding karyopherin α and translocating into thenucleus resides entirely within the HIV-1 Gag proteins. Although otherproteins present in the HIV-1 pre-integration complex (e.g. Vpr, IN, RT)may enhance nuclear translocation, they are not necessary for thisprocess. The results indicate that RT and MA NLS are positioned in closeproximity within the HIV-1 pre-integration complex as CNI-H0294 is verysmall yet seems to bind RT and the MA NLS simultaneously. As MA is madefrom the Gag precursor and RT is made from the Gag-Pol precursor, theratio of RT to MA in the virion is expected to be 1:50 because thetranslational frameshift that leads to synthesis of the Gag-Polprecursor (rather than the Gag precursor) occurs about 2% of the time.Interestingly, about 1-2% of the virion MA protein is phosphorylated andonly these molecules are incorporated into the HIV-1 pre-integrationcomplex. This leads to an important conclusion that there is roughly anequivalent number of RT and MA molecules per HIV-1 pre-integrationcomplex. Given the efficient inactivation of MA NLS by CNI-H0294, mostif not all RT and MA molecules are in close proximity in thepre-integration complex.

The present invention is not to be limited in scope by the specificembodiments described which were intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components were within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

We claim:
 1. A method for inactivating a nuclear localization signal ofa protein comprising contacting the protein with an aryl carbonylcompound that forms stable reversible covalent interactions withneighboring basic amino acid residues of the nuclear localization signalof the protein.
 2. A method for inhibiting importation of a protein intothe nucleus of a cell comprising contacting the protein with an arylcarbonyl compound that forms stable reversible covalent interactionswith neighboring basic amino acid residues of the nuclear localizationsignal of the protein.
 3. A method for targeted inactivation of anuclear localization signal of a protein in a complex comprisingcontacting the protein with an aryl carbonyl compound that: (a)interacts with a molecule in a complex having a specific docking sitewhich is positioned proximately to a nuclear localization signal of aprotein in the complex; and (b) forms stable reversible covalentinteractions with neighboring basic amino acid residues of the nuclearlocalization signal of the protein.
 4. The method of claim 3 wherein thearyl carbonyl compound forms Schiff bases with neighboring lysineresidues of the nuclear localization signal of the protein.
 5. Themethod of claim 3 wherein the aryl carbonyl compound forms stablereversible covalent interactions with neighboring arginine residues ofthe nuclear localization signal of the protein.
 6. A method for targetedinactivation of a nuclear localization signal of a protein comprisingcontacting the protein with a compound according to the formula:

wherein A, independently, =CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃, CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; and P=1 or 2; and

wherein X=NH₂, CH₃ or CH₂CH₃; X′=CH₃ or CH₂CH₃; Y=NH₂, NHCH₃, N(CH₃)₂,1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently,=H, NH₂, NHCH₃, N(CH₃)₂,N⁺(CH₃)₃-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.7. A method for targeted inactivation of a nuclear localization signalof a protein comprising contacting the protein with a compound accordingto the formula:

wherein A=CH₃, CH₂CH₃, COCH₃, COCH₂CH₃, CH₂COCH₃, CH₂COCH₂CH₃,C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; and

wherein X=NH₂, CH₃ or CH₂CH₃; X′=CH₃ or CH₂CH₃; Y=NH₂, NHCH₃, N(CH₃)₂,1-pyrrolidino or 1-piperidino; and Z=H, CH₃ or CH₂CH₃; or

wherein Y′ and Z′, independently, =H, NH₂, NHCH₃, N(CH₃)₂, N⁺(CH₃)₃,1-pyrrolidino or 1-piperidino; Q is N or CH; and salts thereof.
 8. Themethod of claim 3, 4 or 5 wherein the docking site is on the proteinhaving the nuclear localization signal.
 9. The method of claim 3, 4, 5,6 or 7 wherein the protein is derived from a human immunodeficiencyvirus, influenza virus, hepatitis virus, herpes simplex virus,papillomavirus, parvovirus or measles virus.
 10. The method of claim 3wherein the docking site is on the human immunodeficiency virus reversetranscriptase and the nuclear localization signal is in the humanimmunodeficiency virus matrix antigen.
 11. The method of claim 1 whereinthe aryl carbonyl compound forms tandem Schiff bases with neighboringlysine residues of the nuclear localization signal of the protein. 12.The method of claim 1 wherein the aryl carbonyl compound forms residuesof the nuclear localization signal of the protein.
 13. The method ofclaim 3, 4, 5, 6 or 7 wherein the protein is a transcription factor. 14.A method for targeted inactivation of a nuclear localization signal of aprotein, comprising contacting the protein with an aryl carbonylcompound according to the formula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing a sulfur atom; K is 0 or 1; and J is (i) H,(CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight or branchedC₁₋₁₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straight orbranched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, a straightor branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy, whereineach group contains nitrogen or sulfur hetero atoms, or (iii) a mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen or sulfur atom.
 15. A method for targeted inactivation of anuclear localization signal of a protein, comprising contacting theprotein with an aryl carbonyl compound according to the formula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing an oxygen atom; K is 0 or 1; and J is (i)H, (CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight orbranched C₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straightor branched C₁₋₆ alkoxy; (ii) a straight or branched C₂₋₆ alkyl, astraight or branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy,wherein each group contains nitrogen or sulfur hetero atoms, or (iii) amono- or poly-heterocyclic group having 3 to 20 atoms, at least one ofwhich is a nitrogen or sulfur atom.
 16. A method for targetedinactivation of a nuclear localization signal of a protein, comprisingcontacting the protein with an aryl carbonyl compound according to theformula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing a nitrogen atom; K is 0 or 1; and J is (i)H(CH₂)_(n)NH₂, wherein n is an integer from 0 to 6, a straight orbranched C₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straightor branched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, astraight or branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy,wherein each group contains nitrogen or sulfur hetero atoms, or (iii) amono- or poly-heterocyclic group having 3 to 20 atoms, at least one ofwhich is a nitrogen or sulfur atom.
 17. A method for targetedinactivation of a nuclear localization signal of a protein, comprisingcontacting the protein with an aryl carbonyl compound according to theformula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing a carbon atom; K is 0 or 1; and J is (i) H,(CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight or branchedC₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straight orbranched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, a straightor branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy. whereineach group contains nitrogen or sulfur hetero atoms, or (iii) a mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen or sulfur atom.
 18. A method of preventing productive infectionby a virus of a proliferating population of nucleus-containing cells,which comprises the step of contacting the proliferating population ofcells with an aryl carbonyl compound, that forms stable reversiblecovalent interactions with neighboring basic amino acid residues of anuclear localization signal of a protein, preventing importation of acomplex containing viral nucleic acid or viral protein into the nucleusof a cell in the population.
 19. The method of claim 18 which comprisesthe administration of an effective amount of a pharmaceuticalcomposition containing an aryl carbonyl compound wherein the arylcarbonyl compound is 2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyridinium iodide (“Compound No. 2”), or2-amino-4-(3,5-diacetylphenyl) amino-1,6-dimethylpyridinium chloride(CNI-0294; chloride salt of Compound No. 2) as an active ingredient. 20.The method of claim 18, which further comprises the administration of aneffective amount of a pharmaceutical composition containing an arylcarbonyl compound according to the formula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing a sulfur atom; K is 0 or 1; and J is (i) H,(CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight or branchedC₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straight orbranched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, a straightor branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy, whereineach group contains nitrogen or sulfur hetero atoms, or (iii) a mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen or sulfur atom.
 21. The method of claim 18, which furthercomprises the administration of an effective amount of a pharmaceuticalcomposition containing an aryl carbonyl compound according to theformula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing an oxygen atom; K is 0 or 1; and J is (i)H, (CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight orbranched C₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straightor branched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, astraight or branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy,wherein each group contains nitrogen or sulfur hetero atoms, or (iii) amono- or poly-heterocyclic group having 3 to 20 atoms, at least one ofwhich is a nitrogen or sulfur atom.
 22. The method of claim 18, whichfurther comprises the administration of an effective amount of apharmaceutical composition containing an aryl carbonyl compoundaccording to the formula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis a linker group containing a nitrogen atom; K is 0 or 1; and J is (i)H, (CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight orbranched C₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straightor branched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, astraight or branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy,wherein each group contains nitrogen or sulfur hetero atoms, or (iii) amono- or poly-heterocyclic group having 3 to 20 atoms, at least one ofwhich is a nitrogen or sulfur atom.
 23. The method of claim 18, whichfurther comprises the administration of an effective amount of apharmaceutical composition containing an aryl carbonyl compoundaccording to the formula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃,CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; L is a linkergroup containing a carbon atom; K is 0 or 1; and J is (i) H,(CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight or branchedC₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straight orbranched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, a straightor branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy, whereineach group contains nitrogen or sulfur hetero atoms, or (iii) a mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen or sulfur atom.
 24. A method for targeted inactivation of anuclear localization signal of a protein, comprising contacting theprotein with an aryl carbonyl compound according to the formula (I):

wherein A, independently, is CH₃, CH₂CH₃, COH, COCH₃, COCH₂CH₃,CH₂COCH₃, CH₂COCH₂CH₃, C(CH₃)₂COCH₃ or C(CH₃)₂COCH₂CH₃; P is 1 or 2; Lis —SO₂—, —O—, —NH—, —N═, —CH₂— or —CH═; K is 0 or 1; and J is (i) H,(CH₂)_(n)NH₂wherein n is an integer from 0 to 6, a straight or branchedC₁₋₆ alkyl, a straight or branched C₂₋₆ alkenyl or a straight orbranched C₁₋₆ alkoxy; (ii) a straight or branched C₁₋₆ alkyl, a straightor branched C₂₋₆ alkenyl or a straight or branched C₁₋₆ alkoxy, whereineach group contains nitrogen or sulfur hetero atoms, or (iii) a mono- orpoly-heterocyclic group having 3 to 20 atoms, at least one of which is anitrogen or sulfur atom.